Detection of Activation of Endothelial Cells as Surrogate Marker for Angiogenesis

ABSTRACT

Methods, compositions and kits are provided for assessing angiogenesis through sensitive, direct detection of activation of endothelial cells at molecular levels. In general, activation of endothelial cells is detected by measuring the levels of cellular components and their protein complexes participating in a specific angiogenesis signaling pathway in endothelial cells. The methods can be used for assessing status of diseases associated with undesirable angiogenesis, such as the likelihood of developing the disease, presence or absence of the disease, prognosis of the disease and the likelihood of response or resistance to a particular anti-angiogenic therapy. The methods can also be used to guide the design of effective therapeutic regimens targeting a specific angiogenic signaling pathway, as well as in conjunction with therapeutic intervention of diseases or conditions associated with undesirable angiogenesis.

This application claims the benefit of U.S. Provisional Application No.60/625,694, filed Nov. 4, 2004, the contents of which is incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to methods for accessingangiogenesis through detection of the status of endothelial cellactivation and more particularly to methods of determining angiogenicsignal transduction pathway activation at a molecular level, and forusing such status information to select patients responsive topathway-specific drugs, and to rationally design effective therapy withdrugs that specifically modulate angiogenesis signal transductionpathways.

DESCRIPTION OF RELATED ART

Angiogenesis is the fundamental process by which new blood vessels areformed.

The process involves the migration of vascular endothelial cells intotissue, followed by the condensation of such endothelial cells intovessels. Angiogenesis may occur naturally or be induced by an angiogenicagent. The process is essential to a variety of normal body activities,such as reproduction, development and wound repair. Although the processis not completely understood, it involves a complex interplay ofmolecules that stimulate and molecules that inhibit the growth andmigration of endothelial cells, the primary cells of the capillary bloodvessels. Under normal conditions, these molecules appear to maintain themicrovasculature in a quiescent state (i.e., without capillary growth)for prolonged periods which can last for several years or even decades.The turnover time for an endothelial cell is about 1,000 days. Underappropriate conditions, however (e.g., during wound repair), these samecells can undergo rapid proliferation and turnover within a much shorterperiod, and five days is typical under these circumstances. (Folkman etal. (1989) J. Biol. Chem., 267:10931-10934; and Folkman et al. (1987)Science 235: 442-447).

Although angiogenesis is a highly regulated process under normalconditions, many diseases (characterized as “angiogenic diseases”) aredriven by persistent unregulated angiogenesis. In such disease state,unregulated angiogenesis can either cause a particular disease directlyor exacerbate an existing pathological condition. For example, ocularneovascularization has been implicated as the most common cause ofblindness and underlies the pathology of approximately 20 eye diseases.In certain previously existing conditions such as arthritis, newlyformed capillary blood vessels invade the joints and destroy cartilage.In diabetes, new capillaries formed in the retina invade the vitreoushumor, causing bleeding and blindness.

Both the growth and metastasis of solid tumors are alsoangiogenesis-dependent (Folkman (1986) J. Cancer Res. 46:467-473;Folkman (1989) J. Nat. Cancer Inst. 82:4-6; Folkman et al. (1995) “TumorAngiogenesis,” Chapter 10, pp. 206-32, in The Molecular Basis of Cancer,Mendelsohn et al., eds., W. B. Saunders). It has been shown, forexample, that tumors which enlarge to greater than 2 mm. in diametermust obtain their own blood supply and do so by inducing the growth ofnew capillary blood vessels. After these new blood vessels becomeembedded in the tumor, they provide nutrients and growth factorsessential for tumor growth as well as a means for tumor cells to enterthe circulation and metastasize to distant sites, such as liver, lung orbone (Weidner (1991) New Eng. J. Med. 324:1-8).

Cancer cells begin to promote angiogenesis early in tumorigenesis. This“angiogenic switch” (Hanahan et al. (1996) Cell 86:353-364) ischaracterized by oncogene-driven tumour expression of pro-angiogenicproteins (Rak et al. (2000) J. Invest. Dermatol. Symp. Proc. 5:24-33),such as vascular endothelial growth factor (VEGF), basic fibroblastgrowth factor (bFGF), interleukin-8 (IL-8), placenta-like growth factor(PLGF), transforming growth-factor-p (TGF-P), platelet-derivedendothelial growth factor (PEGF), pleiotrophin and others (Relf et al.(1997) Cancer Res. 57:963-969; Carmeliet et al. (1998) Nature394:485-490; and Fukumura et al. (1998) Cell 94:715-725).Tumour-associated hypoxic conditions also activate hypoxia-induciblefactor-1a (HIF-1a) (Carmeliet et al., supra), which promotesupregulation of several angiogeneic factors. Fibroblasts in or near thetumour bed begin to produce pro-angiogenic factors, and tumors alsorecruit progenitor endothelial cells from bone marrow (Shi et al. (1998)Blood 92: 362-367). The angiogenic switch also involves downregulationof angiogenesis suppressor proteins, such as thrombospondin (Dameron etal. (1994) Science 265:1582-1584).

When used as drugs in tumor-bearing animals, natural inhibitors ofangiogenesis can prevent the growth of small tumors (O'Reilly et al.(1994) Cell 79:315-328). Indeed, in some protocols, the application ofsuch inhibitors leads to tumor regression and dormancy even aftercessation of treatment (O'Reilly et al. (1997) Cell, 88, 277-285).Moreover, supplying inhibitors of angiogenesis to certain tumors canpotentiate their response to other therapeutic regimens (e.g.,chemotherapy) (see, e.g., Teischer et al. (1994) Int. J. Cancer 57:920-25).

At present, there is no quantitative method for determining the totalangiogenic output of a patient's tumor burden, so surrogate markers,such as angiogenic proteins present in serum, plasma, and urine. It isrecognized that a significant change in the level of angiogenic proteinsafter initiation of treatment might provide an early indication ofantiangiogenic activity before clinically demonstrable reduction intumor size. Elevated angiogenic growth factors, proteases, andendothelial adhesion molecules have been detected in sera of patientswith malignant diseases (Dirix et al. (1997) Br. J. Cancer 76:238-243).These important promoters of tumor angiogenesis include VEGF, bFGF,urokinase-type plasminogen activator and its soluble receptor (Xu et al.(1997) Hum Pathol 28:206-213), E-selectin and vascular cell adhesionmolecules-1 (VCAM-1) (Banks et al. (1993) Br. J. Cancer 68:122-124), andvon Willebrand's factor (vWF) (Gadducci et al. (1994) Gynecol. Oncol.53:352-356). Of the many mediators of angiogenesis, VEGF and bFGF havebeen frequently measured as potential surrogate markers ofantiangiogenic activity in many clinical studies.

Laser scanning cytometry (LSC) has also been used for quantitativeanalysis of antiangiogenic activity in clinical studies. In LSCmeasurement automated lasers detect individual cells within the mappedregion of tumor biopsy samples based on multicolor immunofluorescencestaining of biomarkers. Each cell is plotted on a scattergram based onits relative fluorescence intensity. LSC-generated scattergrams displaythe percentage of cell populations, for example, apoptotic endothelialcells. Alternative, cellular protein expression levels, e.g.,phosphorylated VEGF receptor-2, may be measured by histogram analysis.See review by Davis et al. (2003) Br. J. Cancer 89:8-14.

Other techniques have been developed to assess changes in microvesseldensity, tumor blood flow, vascular permeability and in some casesmetabolism, such as radiologic techniques including positron emissiontomography (PET), magnetic resonance imaging, dynamic computedtomography and three-dimensional ultrasound. See review by Davis et al.(2003) Br. J. Cancer 89:8-14; and Morgan et al. (2003) J. Clin. Oncol.21: 3955) For example, microvessal density (MVD) which is measured bycounting the distance between vessels is used as a marker forangiogenesis based on the rational that as the distances decrease, theblood vessel density has increased, suggesting that the tumor isextremely angiogenic; and conversely, decease in MVD after therapysuggests that the antiangiogenic therapy is efficacious. Interstitialfluid pressure (IFP) is also used as a marker based on the rational thatas the density of blood vessels increase, the interstitial pressuresincreases. Jain et al. (2004) Nat. Med. 10:145-147.

Recently ex vivo analyses of isolated peripheral blood cells have beenused to monitor surrogate markers of antiangiogenic activity. In onestudy, a cytokine release assay was used to measure the effort of MM1270(a matrix metalloproteinase inhibitor, previously termed CGS270231,Eatock et al. (1999) J. Clin. Oncol. 18:209a) on release of tumornecrosis factor-a from ex vivo stimulated peripheral blood cells (Levittet al. (2001) Clin. Cancer Res. 7:1912-1922). Flow cytometry has alsobeen used to quantify activated circulating endothelial cells from theperipheral blood of cancer patients in an effort to assess the effectsof antiangiogenic activity (Mancuso et al. (2001) Blood 97:3658-3661;and Monestiroli et al. (2001) Cancer Res. 61:4341-4344). Mancuso et al.found that resting and activated endothelial cells are increased innewly diagnosed cancer patients and decline after cure; and Monestiroliet al. demonstrated that there was a strong correlation betweencirculating endothelial cells (CEC) and tumor volume and between CEC andtumor-generated VEGF.

There are various disadvantages associated with above approaches toassessing tumor angiogenesis. Although bFGF and VEGF levels have beendeveloped as useful surrogate markers for determining the response tothalidomide or IFN-α therapy, there is an urgent need for surrogatemarkers to determine efficacy of other types of anti-angiogenictherapies. For most tumors, it is unlikely that quantification ofcirculating factors will serve as useful surrogate markers. Tumors cangenerate various positive and negative regulators of angiogenesis.Bergers et al. (2003) Nature Review 3:401-410. To determine whether atumor is growing or regressing, it would be necessary to quantify theplasma or urine concentration of all of these mediators, which is notfeasible at present. Quantification of microvessel density, althoughvaluable as a predictor of future risk of metastasis or mortality, hasnot proven to be a useful indicator of efficacy of currentanti-angiogenic therapy. Kerbel et al. (2002) Nature Review 2:727-739.In addition, the above methods do not directly measure activation ofendothelial cells and are difficult to employ in practice.

SUMMARY OF THE INVENTION

The present invention provides an innovative approach to assessingangiogenesis through sensitive, direct detection of activation of cellsthat contribute to or promote angiogenesis, e.g., endothelial cells,stroma cells or tumor cells, ata molecular level. In general, activationof such cells is detected by measuring the levels of cellular componentsand their protein complexes participating in a specific angiogenesissignaling pathway in these cells. Compared with the currently availabletechniques for assessing angiogenesis, the methods provided in thepresent invention can sensitively and conveniently measure angiogenesisand detect the earliest indication of angiogenesis. Because theinventive methods can be employed to directly monitor a specificangiogenic signaling pathway, a more rational, patient-tailored therapycan be developed by targeting the specific pathway. Moreover, theinventive methods can also be used for assessing status of diseasesassociated with aberrant angiogenesis, such as the likelihood ofdeveloping the disease, presence or absence of the disease, prognosis ofthe disease and the likelihood of response or resistance to a particularanti-angiogenic therapy.

In one aspect, the present invention provides methods for detectingactivation of endothelial cells in a test sample. The methods comprisethe step of measuring the level of a protein complex in the endothelialcells in a test sample, wherein the protein complex is formed between afirst cellular component and a second cellular component that arecellular components in an angiogenesis signaling pathway. A differencein the level of the protein complex relative to the level of the proteincomplex in a reference sample detects activation of endothelial cells inthe test sample.

The protein complexes is formed between the first and second cellularcomponents. The first and second cellular components can be any knowncellular components in an angiogenesis signaling pathway, including butnot limited to VEGFR, Nrp, heparin sulphate, VE-cadherin, Tie, VEGF,PlGF, PDGFR, EphA, EphB, Flt, FGFR, Stat, BAD, RSK, P13K, FAK, Src,P70S6K, SHC, SHC, Akt, Erk, JNK, P38, and MEK. In preferred embodiments,the protein complex is VEGFR1 homodimers, VEGFR2 homodimers,VEGFR1-VEGFR2 heterodimers, VEGFR2-VEGFR3 heterodimers, VEGFR2-SHCcomplexes, or VEGFR3-SHC complexes.

Test samples for use in the present invention may come from a widevariety of sources, including cell cultures, animal or plant tissues,patient biopsies, patient blood sample etc. In some embodiments, thetest sample is a blood sample or a fixed tissue sample. In someembodiments, the test sample is obtained from an individual who issuspected of having a disease associated with undesirable angiogenesis,and wherein detecting activation of endothelial cells in the test sampleindicates that the individual has the disease.

In some embodiments, the test sample may contain circulating endothelialcells, circulating endothelial cell progenitors or tumor endothelium. Incertain embodiments, the methods further comprise isolating circulatingendothelial cells or circulating endothelial cell progenitors. Inpreferred embodiments, circulating endothelial cells or circulatingendothelial cell progenitors are isolated by immunomagnetic isolation.

The level of a protein complex in the endothelial cells can be measuredby any techniques known to those of skill in the art. For example,immunoaffinity-based methods, cross-linking assays, or fluorescenceresonance energy transfer can be utilized.

In preferred embodiments, assays using releasable molecular tags areused to detect protein complexes in the endothelial cells. In suchembodiments, the step of measuring the protein complex in theendothelial cells comprises mixing (i) the test sample; (ii) a cleavingprobe, which is capable of binding the first cellular component and hasa cleavage-inducing moiety with an effective proximity; and (iii) one ormore binding compounds, wherein each of the binding compounds is capableof binding the first or second cellular component and wherein each ofthe one or more binding compounds has one or more molecular tags eachattached thereto by a cleavable linkage; wherein cleavage of thecleavable linkage(s) within the effective proximity of thecleaving-inducing moiety of the cleaving probe releases the moleculartag(s), wherein detecting the released molecular tag(s) provides ameasurement of the protein complex. The cleaving probes, bindingcompounds, cleavage-inducing moiety and cleavable linkages are describedin detail herein.

In some embodiments, the methods further comprise separating thereleased molecular tag(s). In other embodiments. The methods furthercomprise measuring the level of an effector protein in the angiogenesissignaling pathway that has a post-translational modification site in theendothelial cells in the test sample. The effector proteins aredescribed herein.

In another aspect, the present invention provides methods fordetermining a disease status of an individual who has or likely has adisease associated with undesirable angiogenesis. The methods comprisemeasuring the level of a protein complex in the endothelial cells in atest sample from an individual, wherein the protein complex is formedbetween a first cellular component and a second cellular component thatare cellular components in an angiogenesis signaling pathway. Adifference between the level of the protein complex in the test sampleand a reference level of the protein complex indicates the diseasestatus of the patient.

In another aspect, the present invention provides methods for screeningpatients to determine the likelihood that a patient will respond totreatment by an anti-angiogenic agent. The methods comprise measuringthe level of a protein complex in the endothelial cells in a test samplefrom a patient, wherein the protein complex is formed between a firstcellular component and a second cellular component that are cellularcomponents in an angiogenesis signaling pathway. An increase in thelevel of the protein complex in the test sample from the patientrelative to a reference level characteristic of normal endothelial cellsindicates that the likelihood that the patient will respond to treatmentby an anti-angiogenic agent.

In another aspect, the present invention provides methods fordetermining whether a patient will respond to treatment by ananti-angiogenic agent. The methods comprise measuring the level of aprotein complex in the endothelial cells in a test sample from a patientwho has been treated with an anti-angiogenic agent, wherein the proteincomplex is formed between a first cellular component and a secondcellular component that are cellular components in an angiogenesissignaling pathway. An increase in the level of the protein complex inthe vascular (or CEC/CECP) endothelial cells from the test samplerelative to a reference level of the protein complex in endothelialcells in the patient prior to the treatment, indicates that the patientis likely to respond to the treatment by the anti-angiogenic agent.

In another aspect, the present invention provides methods fordetermining whether a patient has developed resistance to treatment ofan anti-angiogenic agent. The methods comprise measuring the level of aprotein complex in the endothelial cells in a test sample from a patientwho has been treated with an anti-angiogenic agent, wherein the proteincomplex is formed between a first cellular component and a secondcellular component that are cellular components in an angiogenesissignaling pathway. An increase in the level of the protein complex inthe endothelial cells from the test sample relative to a reference levelof the protein complex in the endothelial cells in the patient prior totreatment, indicates that the patient has likely developed resistance tothe treatment of the anti-angiogenic agent.

In another aspect, the present invention provides methods for detectingactivation of endothelial cells in a test sample. The methods comprisemeasuring in a test sample the levels of two or more different cellularcomponents that participate in one or more angiogenesis signalingpathways. A difference in the levels of the two or more differentcellular components relative to reference levels of the two or moredifferent cellular components, indicates activation of endothelial cellsin a test sample.

The two or more cellular components can be any known cellular componentsthat participate in one or more angiogenesis signaling pathway and aredescribed in detail herein.

In some embodiments, the test sample may contain circulating endothelialcells, circulating endothelial cell progenitors or tumor endothelium. Incertain embodiments, the methods further comprise isolating circulatingendothelial cells or circulating endothelial cell progenitors. Inpreferred embodiments, circulating endothelial cells or circulatingendothelial cell progenitors are isolated by immunomagnetic isolation.

The level of the two or more cellular components can be measured by anytechniques known to those of skill in the art. For example,immunoaffinity-based methods, cross-linking assays, or fluorescenceresonance energy transfer can be utilized.

In preferred embodiments, assays using releasable molecular tags areused to detect the level of two more cellular components in theendothelial cells. In such embodiments, the step of measuring the levelsof two or more different cellular components in the endothelial cellscomprises mixing (i) the test sample; (ii) a cleaving probe, which iscapable of binding one of the two or more cellular components and has acleavage-inducing moiety with an effective proximity; and (iii) one ormore binding compounds, wherein each of the two or more cellularcomponents is bound by at least one member of the one or more bindingcompounds, and wherein each of the binding compounds has one or moremolecular tags each attached thereto by a cleavable linkage; whereincleavage of the cleavable linkage(s) within the effective proximity ofthe cleaving-inducing moiety of the cleaving probe releases themolecular tag(s), wherein detecting the released molecular tag(s)provides a measurement of the levels of two or more different cellularcomponents in the endothelial cells. The cleaving probes, bindingcompounds, cleavage-inducing moiety and cleavable linkages are describedin detail herein.

In some embodiments, the methods further comprise separating thereleased molecular tag(s). In other embodiments, The methods furthercomprise measuring the level of an effector protein in the angiogenesissignaling pathway that has a post-translational modification site in theendothelial cells in the test sample. The effector proteins aredescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the role angiogenesis plays in the growth andmetastasis of a small localized tumor.

FIG. 2 illustrates exemplary families of receptor tyrosine kinase (RTK).

FIG. 3 illustrates exemplary signal transduction pathways of VEGFR2 inresponse to VEGF.

FIGS. 4A-4F illustrate diagrammatically the use of releasable moleculartags to measure receptor dimer populations.

FIGS. 4G-4H illustrate diagrammatically the use of releasable moleculartags to measure cell surface receptor complexes in fixed tissuespecimens.

FIGS. 5A-5E illustrate diagrammatically methods for attaching moleculartags to antibodies.

FIG. 6 illustrates an embodiment for measuring relative amounts ofreceptor dimers containing a common component receptor.

FIG. 7A illustrates diagrammatically an embodiment for measuringVEGFR2-VEGFR2 homodimers and VEGFR2 phosphorylation.

FIGS. 7B-D show measurements of VEGFR2-VEGFR2 homodimers and VEGFR2phosphorylation in HUVEC cells in response to VEGF treatment.

FIGS. 8A-B show measurements of Tie2-Tie2 homodimer and Tie2 in HUVECcells in response to Ang-1 treatment.

FIGS. 9A-B show measurements of VEGFR2 in HUVEC cells isolated from amixture U-937 and HUVEC cells.

FIGS. 10A, 10B, and 10C show measurements of phosphorylation of Erk,RSK, and BAD respectively in HUVEC cells in response to VEGF treatment.

FIGS. 11A and 11B show formulas for NHS esters of various molecular tagsused with the invention.

FIG. 12 shows measurement of VEGFR2-VEGFR2 homodimers in HUVEC celllysate and in formalin-fixed paraffin embedded (“FFPE”) samples.

FIG. 13A shows specificity of the cleaving probe to human VEGFR2.

FIG. 13B shows results of xenograph screening for VEGFR2-VEGFR2homodimers.

FIG. 14 shows measurement of VEGFR2-VEGFR2 homodimers in various lungcancer tissue and normal tissue samples.

DEFINITIONS

“Akt protein” means a human protein that is a member of the set ofPKBa/Akt1, PKBb/Akt2, PKBg/Akt3, PKBg-1, and proteins havingsubstantially identical amino acid sequences thereof, and that hasprotein kinase activity whenever phosphorylated by a PI3K protein. Inone aspect, an Akt protein has kinase activity whenever either or bothof a tyrosine at a location number from 305 to 310 is phosphorylated anda serine at location number from 470 to 475 is phosphorylated. Aktproteins are described under various NCBI accession numbers, includingNP_(—)005154, and in Nicholson et al., Cellular Signalling, 14: 381-395(2002); Kandel et al., Exp. Cell. Res., 253: 210-229 (1999); and likereferences, all of which are incorporated herein by references.

“Effector protein” means an intracellular protein that is a component ofa signal transduction pathway and that may be chemically alteredresulting in the acquisition or loss of an activity or property. Suchchemical alteration may include any of the post-translationalmodifications listed below as well as processing by proteinases. In oneaspect, effector proteins are chemically modified by phosphorylation andacquire protein kinase activity as a result of such phosphorylation. Inanother aspect, effector proteins are chemically modified byphosphorylation and lose protein kinase activity as a result of suchphosphorylation. In another aspect, effector proteins are chemicallymodified by phosphorylation and lose the ability to form stablecomplexes with particular proteins as a result of such phosphorylation.Exemplary effector proteins include, but are not limited to, Aktproteins, Erk proteins, p38 proteins, and Jnk proteins. In regard topost-translational modifications of effector proteins, an effectorprotein may have one or more sites, referred to herein as a“post-translational modification site,” which are characteristic aminoacids of the effector protein where a post-translational modificationmay be attached or removed in the course of a signal transduction event.

“MAPK protein” means a human protein of the set of Erk1 proteins, Erk2proteins (collectively referred to as Erk1/2 proteins), p38 proteins,Jnk proteins, and proteins having amino acid sequences substantiallyidentical thereto.

“Phosphatidylinositol 3 kinase protein,” or equivalently a “PI3Kprotein,” means a human intracellular protein of the set of humanproteins describe under NCBI accession numbers NP_(—)852664,NP_(—)852556, and NP_(—)852665, and proteins having amino acid sequencessubstantially identical thereto.

“Substantially identical” in reference to proteins or amino acidsequences of proteins in a family of related proteins that are beingcompared means either that one protein has an amino acid sequence thatis at least fifty percent identical to the other protein or that oneprotein is an isoform or splice variant of the same gene as the otherprotein. In one aspect, substantially identical means one protein, oramino acid sequence thereof, is at least eighty percent identical to theother protein, or amino acid sequence thereof.

“Antibody” means an immunoglobulin that specifically binds to, and isthereby defined as complementary with, a particular spatial and polarorganization of another molecule. The antibody can be monoclonal orpolyclonal and can be prepared by techniques that are well known in theart such as immunization of a host and collection of sera (polyclonal)or by preparing continuous hybrid cell lines and collecting the secretedprotein (monoclonal), or by cloning and expressing nucleotide sequencesor mutagenized versions thereof coding at least for the amino acidsequences required for specific binding of natural antibodies.Antibodies may include a complete immunoglobulin or fragment thereof,which immunoglobulins include the various classes and isotypes, such asIgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereofmay include Fab, Fv and F(ab′)2, Fab′, and the like. In addition,aggregates, polymers, and conjugates of immunoglobulins or theirfragments can be used where appropriate so long as binding affinity fora particular polypeptide is maintained. Guidance in the production andselection of antibodies for use in immunoassays, including such assaysemploying releasable molecular tag (as described below) can be found inreadily available texts and manuals, e.g. Harlow and Lane, Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory Press, New York, 1988);Howard and Bethell, Basic Methods in Antibody Production andCharacterization (CRC Press, 2001); Wild, editor. The ImmunoassayHandbook (Stockton Press, New York, 1994), and the like.

“Antibody binding composition” means a molecule or a complex ofmolecules that comprises one or more antibodies, or fragments thereof,and derives its binding specificity from such antibody or antibodyfragment. Antibody binding compositions include, but are not limited to,(i) antibody pairs in which a first antibody binds specifically to atarget molecule and a second antibody binds specifically to a constantregion of the first antibody; a biotinylated antibody that bindsspecifically to a target molecule and a streptavidin protein, whichprotein is derivatized with moieties such as molecular tags orphotosensitizers, or the like, via a biotin moiety; (ii) antibodiesspecific for a target molecule and conjugated to a polymer, such asdextran, which, in turn, is derivatized with moieties such as moleculartags or photosensitizers, either directly by covalent bonds orindirectly via streptavidin-biotin linkages; (iii) antibodies specificfor a target molecule and conjugated to a bead, or microbead, or othersolid phase support, which, in turn, is derivatized either directly orindirectly with moieties such as molecular tags or photosensitizers, orpolymers containing the latter.

“Antigenic determinant,” or “epitope” means a site on the surface of amolecule, usually a protein, to which a single antibody molecule binds;generally a protein has several or many different antigenic determinantsand reacts with antibodies of many different specificities. A preferredantigenic determinant is a phosphorylation site of a protein.

“Binding moiety” means any molecule to which molecular tags can bedirectly or indirectly attached that is capable of specifically bindingto an analyte. Binding moieties include, but are not limited to,antibodies, antibody binding compositions, peptides, proteins, nucleicacids, and organic molecules having a molecular weight of up to 1000daltons and consisting of atoms selected from the group consisting ofhydrogen, carbon, oxygen, nitrogen, sulfur, and phosphorus. Preferably,binding moieties are antibodies or antibody binding compositions.

“Complex” as used herein means an assemblage or aggregate of moleculesin direct or indirect contact with one another. In one aspect,“contact,” or more particularly, “direct contact” in reference to acomplex of molecules, or in reference to specificity or specificbinding, means two or more molecules are close enough so that attractivenoncovalent interactions, such as Van der Waal forces, hydrogen bonding,ionic and hydrophobic interactions, and the like, dominate theinteraction of the molecules. In such an aspect, a complex of moleculesis stable in that under assay conditions the complex isthermodynamically more favorable than a non-aggregated, ornon-complexed, state of its component molecules. As used herein,“complex” usually refers to a stable aggregate of two or more proteins,and is equivalently referred to as a “protein-protein complex.” Mosttypically, a “complex” refers to a stable aggregate of two proteins. Asused herein, an “intracellular complex” or “intracellularprotein-protein complex,” refers to a complex of proteins normally foundin the cytoplasm or nucleus of a biological cell, and may includecomplexes of one or more intracellular proteins and a surface membranereceptor. In another aspect, a complex is a stable aggregate comprisingtwo proteins, or from 2 to 4 proteins, or from 2 to 6 proteins. As usedherein, a “signaling complex” is an intracellular protein-proteincomplex that is a component of a signaling pathway.

“Dimer” in reference to cell surface membrane receptors means a complexof two or more membrane-bound receptor proteins that may be the same ordifferent. Dimers of identical receptors are referred to as “homodimers”and dimers of different receptors are referred to as “heterodimers.”Dimers usually consist of two receptors in contact with one another.Dimers may be created in a cell surface membrane by passive processes,such as Van der Waal interactions, and the like, as described above inthe definition of “complex,” or dimers may be created by activeprocesses, such as by ligand-induced dimerization, covalent linkages,interaction with intracellular components, or the like, e.g.Schlessinger, Cell, 103: 211-225 (2000).

“Disease status” includes, but is not limited to, the followingfeatures: likelihood of contracting a disease, presence or absence of adisease, prognosis of disease severity, and likelihood that a patientwill respond to treatment by a particular therapeutic agent thatmodulate an angiogenesis signal transduction pathway. In regard tocancer, “disease status” further includes detection of precancerous orcancerous cells or tissues, the selection of patients that are likely torespond to treatment by a therapeutic agent that inhibits angiogenesisof tumors, and the ameliorative effects of treatment with suchtherapeutic agents.

“Her receptor” is a receptor protein tyrosine kinase which belongs tothe ErbB receptor family and includes EGFR (“Her1”), ErbB2 (“Her2”),ErbB3 (“Her3”) and ErbB4 (“Her4”) receptors. The Her receptor generallycomprises an extracellular domain, which may bind an ErbB ligand; alipophilic transmembrane domain; a conserved intracellular tyrosinekinase domain; and a carboxyl-terminal signaling domain harboringseveral tyrosine residues which can be phosphorylated. The Her receptormay be a native sequence ErbB receptor or an amino acid sequence variantthereof. Preferably the ErbB receptor is native sequence human ErbBreceptor. In one aspect, the Her receptor includes truncated versions ofHer receptors, including but not limited to, EGFRvIII and p95Her2,disclosed in Chu et al., Biochem. J., 324: 855-861 (1997); Xia et al.,Oncogene, 23: 646-653 (2004); and the like. As used herein, a “Herreceptor complex” is a complex or receptor complex containing at leastone Her receptor.

The terms “ErbB1”, “epidermal growth factor receptor” and “EGFR” and“Her1” are used interchangeably herein and refer to native sequence EGFRas disclosed, for example, in Carpenter et al. Ann. Rev. Biochem.56:881-914 (1987), including variants thereof (e.g. a deletion mutantEGFR as in Humphrey et al. PNAS (USA) 87:4207-4211 (1990)). erbB1 refersto the gene encoding the EGFR protein product. Examples of antibodieswhich bind to EGFR include MAb 579 (ATCC CRL RB 8506), MAb 455 (ATCC CRLHB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S.Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such aschimerized 225 (C225) and reshaped human 225 (H225) (see, WO 96/40210,Imclone Systems Inc.).

“Her2”, “ErbB2” “c-Erb-B2” are used interchangeably. Unless indicatedotherwise, the terms “ErbB2” “c-Erb-B2” and “Her2” when used hereinrefer to the human protein. The human ErbB2 gene and ErbB2 protein are,for example, described in Semba et al., PNAS (USA) 82:6497-650 (1985)andYamamoto et al. Nature 319:230-234 (1986) (Genebank accession numberX03363). Examples of antibodies that specifically bind to Her2 aredisclosed in U.S. Pat. Nos. 5,677,171; 5,772,997; Fendly et al., CancerRes., 50: 1550-1558 (1990); and the like.

“ErbB3” and“Her3” refer to the receptor polypeptide as disclosed, forexample, in U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus etal. PNAS (USA) 86:9193-9197 (1989), including variants thereof. Examplesof antibodies which bind Her3 are described in U.S. Pat. No. 5,968,511,e.g. the 8B8 antibody (ATCC HB 12070).

The terms “ErbB4” and “Her4” herein refer to the receptor polypeptide asdisclosed, for example, in EP Pat Appin No 599,274; Plowman et al.,Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman et al.,Nature, 366:473-475 (1993), including variants thereof such as the Her4isoforms disclosed in WO 99/19488.

“Insulin-like growth factor-1 receptor” or “IGF-1R” means a humanreceptor tyrosine kinase substantially identical to those disclosed inUllrich et al., EMBO J., 5: 2503-2512 (1986) or Steele-Perkins et al.,J. Biol. Chem., 263: 11486-11492 (1988).

“Isolated” in reference to a polypeptide or protein means substantiallyseparated from the components of its natural environment. Preferably, anisolated polypeptide or protein is a composition that consists of atleast eighty percent of the polypeptide or protein identified bysequence on a weight basis as compared to components of its naturalenvironment; more preferably, such composition consists of at leastninety-five percent of the polypeptide or protein identified by sequenceon a weight basis as compared to components of its natural environment;and still more preferably, such composition consists of at leastninety-nine percent of the polypeptide or protein identified by sequenceon a weight basis as compared to components of its natural environment.Most preferably, an isolated polypeptide or protein is a homogeneouscomposition that can be resolved as a single spot after conventionalseparation by two-dimensional gel electrophoresis based on molecularweight and isoelectric point. Protocols for such analysis byconventional two-dimensional gel electrophoresis are well known to oneof ordinary skill in the art, e.g. Hames and Rickwood, Editors, GelElectrophoresis of Proteins: A Practical Approach (IRL Press, Oxford,1981); Scopes, Protein Purification (Springer-Verlag, New York, 1982);Rabilloud, Editor, Proteome Research: Two-Dimensional GelElectrophoresis and Identification Methods (Springer-Verlag, Berlin,2000).

“Kit” refers to any delivery system for delivering materials or reagentsfor carrying out a method of the invention. In the context of reactionassays, such delivery systems include systems that allow for thestorage, transport, or delivery of reaction reagents (e.g., probes,enzymes, etc. in the appropriate containers) and/or supporting materials(e.g., buffers, written instructions for performing the assay etc.) fromone location to another. For example, kits include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. Such contents may be delivered to theintended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a secondcontainer contains probes.

“Pathway-specific drug” means a drug designed to inhibit or block asignal transduction pathway by interacting with, or targeting, acomponent of the pathway to inhibit or block a protein-proteininteraction, such as receptor dimerization, or to inhibit or block anenzymatic activity, such as a kinase activity or a phosphatase activity.Tables 1 and 2 (shown in Section 5 entitled “Examples of AntiangiogenicAgents” below) list exemplary angiogenesis inhibitors that targetvarious angiogenesis pathways.

“Percent identical,” or like term, used in respect of the comparison ofa reference sequence and another sequence (i.e. a “candidate” sequence)means that in an optimal alignment between the two sequences, thecandidate sequence is identical to the reference sequence in a number ofsubunit positions equivalent to the indicated percentage, the subunitsbeing nucleotides for polynucleotide comparisons or amino acids forpolypeptide comparisons. As used herein, an “optimal alignment” ofsequences being compared is one that maximizes matches between subunitsand minimizes the number of gaps employed in constructing an alignment.Percent identities may be determined with commercially availableimplementations of algorithms described by Needleman and Wunsch, J. Mol.Biol., 48: 443-453 (1970)(“GAP” program of Wisconsin Sequence AnalysisPackage, Genetics Computer Group, Madison, Wis.). Other softwarepackages in the art for constructing alignments and calculatingpercentage identity or other measures of similarity include the“BestFit” program, based on the algorithm of Smith and Waterman,Advances in Applied Mathematics, 2: 482-489 (1981) (Wisconsin SequenceAnalysis Package, Genetics Computer Group, Madison, Wis.). In otherwords, for example, to obtain a polypeptide having an amino acidsequence at least 95 percent identical to a reference amino acidsequence, up to five percent of the amino acid residues in the referencesequence many be deleted or substituted with another amino acid, or anumber of amino acids up to five percent of the total amino acidresidues in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence many occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence of in one or morecontiguous groups with in the references sequence. It is understood thatin making comparisons with reference sequences of the invention thatcandidate sequence may be a component or segment of a larger polypeptideor polynucleotide and that such comparisons for the purpose computingpercentage identity is to be carried out with respect to the relevantcomponent or segment.

“Platelet-derived growth factor receptor” or “PDGFR” means a humanreceptor tyrosine kinase protein that is substantially identical toPDGFRα or PDGFRβ, or variants thereof, described in Heldin et al.,Physiological Reviews, 79: 1283-1316 (1999). In one aspect, theinvention includes determining the status of cancers, pre-cancerousconditions, fibrotic or sclerotic conditions by measuring one or moredimers of the following group: PDGFRα homodimers, PDGFRβ homodimers, andPDGFRα-PDGFRβ heterodimers. In particular, fibrotic conditions includelung or kidney fibrosis, and sclerotic conditions includeatherosclerosis. Cancers include, but are not limited to, breast cancer,colorectal carcinoma, glioblastoma, and ovarian carcinoma. Reference to“PDGFR” alone is understood to mean “PDGFRα” or “PDGFRβ.” PDGFRs aredisclosed in Heldin et al., Physiological Reviews, 79: 1283-1316 (1999),and in various NCBI accession numbers.

“Polypeptide” refers to a class of compounds composed of amino acidresidues chemically bonded together by amide linkages with eliminationof water between the carboxy group of one amino acid and the amino groupof another amino acid. A polypeptide is a polymer of amino acidresidues, which may contain a large number of such residues. Peptidesare similar to polypeptides, except that, generally, they are comprisedof a lesser number of amino acids. Peptides are sometimes referred to asoligopeptides. There is no clear-cut distinction between polypeptidesand peptides. For convenience, in this disclosure and claims, the term“polypeptide” will be used to refer generally to peptides andpolypeptides. The amino acid residues may be natural or synthetic.

“Protein” refers to a polypeptide, usually synthesized by a biologicalcell, folded into a defined three-dimensional structure. Proteins aregenerally from about 5,000 to about 5,000,000 or more in molecularweight, more usually from about 5,000 to about 1,000,000 molecularweight, and may include post-translational modifications, suchacetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, farnesylation,demethylation, formation of covalent cross-links, formation of cystine,formation of pyroglutamate, formylation, gamma-carboxylation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, phosphorylation, prenylation,racemization, selenoylation, sulfation, and ubiquitination, e.g. Wold,F., Post-translational Protein Modifications: Perspectives andProspects, pgs. 1-12 in Post-translational Covalent Modification ofProteins, B. C. Johnson, Ed., Academic Press, New York, 1983. In oneaspect, post-translational modifications are usually phosphorylations ofproteins that are components of a signaling pathway. Proteins include,by way of illustration and not limitation, cytokines or interleukins,enzymes such as, e.g., kinases, proteases, galactosidases and so forth,protamines, histones, albumins, immunoglobulins, scleroproteins,phosphoproteins, mucoproteins, chromoproteins, lipoproteins,nucleoproteins, glycoproteins, T-cell receptors, proteoglycans, and thelike.

“Reference sample” means one or more cell, xenograft, or tissue samplesthat are representative of a normal or non-diseased state to whichmeasurements on patient samples are compared to determine whether areceptor complex is present in excess or is present in reduced amount inthe patient sample. The nature of the reference sample is a matter ofdesign choice for a particular assay and may be derived or determinedfrom normal tissue of the patient him- or herself, or from tissues froma population of healthy individuals. Preferably, values relating toamounts of receptor complexes in reference samples are obtained underessentially identical experimental conditions as corresponding valuesfor patient samples being tested. Reference samples may be from the samekind of tissue as that the patient sample, or it may be from differenttissue types, and the population from which reference samples areobtained may be selected for characteristics that match those of thepatient, such as age, sex, race, and the like. Typically, in assays ofthe invention, amounts of receptor complexes on patient samples arecompared to corresponding values of reference samples that have beenpreviously tabulated and are provided as average ranges, average valueswith standard deviations, or like representations.

“Receptor complex” means a complex that comprises at least one cellsurface membrane receptor. Receptor complexes may include a dimer ofcell surface membrane receptors, or one or more intracellular proteins,such as adaptor proteins, that form links in the various signalingpathways.

“Receptor tyrosine kinase,” or “RTK,” means a human receptor proteinhaving intracellular kinase activity and being selected from the RTKfamily of proteins described in Schlessinger, Cell, 103: 211-225 (2000);and Blume-Jensen and Hunter (cited above). “Receptor tyrosine kinasedimer” means a complex in a cell surface membrane comprising tworeceptor tyrosine kinase proteins. In some aspects, a receptor tyrosinekinase dimer may comprise two covalently linked receptor tyrosine kinaseproteins.

“Response index” means a number that is a value of a function thatdepends on one or more measured quantities or arithmetic expressionsthereof. “Function” as used herein has its conventional mathematicaldefinition. The measured quantities include the amounts of cell surfacereceptors, cell surface receptor complexes, signaling complexes, andpost-translational modifications thereof. The function and arithmeticexpressions employed depend on several factors including, but notlimited to, the pathway-specific drug being considered, the type ofdisease, genotype of a patient, and the like.

“Sample” or “tissue sample” or “patient sample” or “patient cell ortissue sample” or “specimen” each means a collection of similar cellsobtained from a tissue of a subject or patient. The source of the tissuesample may be solid tissue as from a fresh, frozen and/or preservedorgan or tissue sample or biopsy or aspirate; blood or any bloodconstituents; bodily fluids such as cerebral spinal fluid, amnioticfluid, peritoneal fluid, or interstitial fluid; or cells from any timein gestation or development of the subject. The tissue sample maycontain compounds which are not naturally intermixed with the tissue innature such as preservatives, anticoagulants, buffers, fixatives,nutrients, antibiotics, or the like. In one aspect of the invention,tissue samples or patient samples are fixed, particularly conventionalformalin-fixed paraffin-embedded samples. Such samples are typicallyused in an assay for receptor complexes in the form of thin sections,e.g. 3-10 μm thick, of fixed tissue mounted on a microscope slide, orequivalent surface. Such samples also typically undergo a conventionalre-hydration procedure, and optionally, an antigen retrieval procedureas a part of, or preliminary to, assay measurements.

“Separation profile” in reference to the separation of molecular tagsmeans a chart, graph, curve, bar graph, or other representation ofsignal intensity data versus a parameter related to the molecular tags,such as retention time, mass, or the like, that provides a readout, ormeasure, of the number of molecular tags of each type produced in anassay. A separation profile may be an electropherogram, a chromatogram,an electrochromatogram, a mass spectrogram, or like graphicalrepresentation of data depending on the separation technique employed. A“peak” or a “band” or a “zone” in reference to a separation profilemeans a region where a separated compound is concentrated. There may bemultiple separation profiles for a single assay if, for example,different molecular tags have different fluorescent labels havingdistinct emission spectra and data is collected and recorded at multiplewavelengths. In one aspect, released molecular tags are separated bydifferences in electrophoretic mobility to form an electropherogramwherein different molecular tags correspond to distinct peaks on theelectropherogram. A measure of the distinctness, or lack of overlap, ofadjacent peaks in an electropherogram is “electrophoretic resolution,”which may be taken as the distance between adjacent peak maximumsdivided by four times the larger of the two standard deviations of thepeaks. Preferably, adjacent peaks have a resolution of at least 1.0, andmore preferably, at least 1.5, and most preferably, at least 2.0. In agiven separation and detection system, the desired resolution may beobtained by selecting a plurality of molecular tags whose members haveelectrophoretic mobilities that differ by at least a peak-resolvingamount, such quantity depending on several factors well known to thoseof ordinary skill, including signal detection system, nature of thefluorescent moieties, the diffusion coefficients of the tags, thepresence or absence of sieving matrices, nature of the electrophoreticapparatus, e.g. presence or absence of channels, length of separationchannels, and the like. Electropherograms may be analyzed to associatefeatures in the data with the presence, absence, or quantities ofmolecular tags using analysis programs, such as disclosed in Williams etal., U.S. patent publication 2003/0170734 A1.

“SHC” (standing for “Src homology 2/a-collagen-related”) means any oneof a family of adaptor proteins (66, 52, and 46 kDalton) in RTKsignaling pathways substantially identical to those described in Pelicciet al., Cell, 70: 93-104 (1992). In one aspect, SHC means the humanversions of such adaptor proteins.

“Signaling pathway” or “signal transduction pathway” means a series ofmolecular events usually beginning with the interaction of cell surfacereceptor and/or receptor dimer with an extracellular ligand or with thebinding of an intracellular molecule to a phosphorylated site of a cellsurface receptor. Such beginning event then triggers a series of furthermolecular interactions or events, wherein the series of such events orinteractions results in a regulation of gene expression, for example, byregulation of transcription in the nucleus of a cell, or by regulationof the processing or translation of mRNA transcripts.

“Specific” or “specificity” in reference to the binding of one moleculeto another molecule, such as a binding compound, or probe, for a targetanalyte or complex, means the recognition, contact, and formation of astable complex between the probe and target, together with substantiallyless recognition, contact, or complex formation of the probe with othermolecules. In one aspect, “specific” in reference to the binding of afirst molecule to a second molecule means that to the extent the firstmolecule recognizes and forms a complex with another molecules in areaction or sample, it forms the largest number of the complexes withthe second molecule. In one aspect, this largest number is at leastfifty percent of all such complexes form by the first molecule.Generally, molecules involved in a specific binding event have areas ontheir surfaces or in cavities giving rise to specific recognitionbetween the molecules binding to each other. Examples of specificbinding include antibody-antigen interactions, enzyme-substrateinteractions, formation of duplexes or triplexes among polynucleotidesand/or oligonucleotides, receptor-ligand interactions, and the like.

“Spectrally resolvable” in reference to a plurality of fluorescentlabels means that the fluorescent emission bands of the labels aresufficiently distinct, i.e. sufficiently non-overlapping, that moleculartags to which the respective labels are attached can be distinguished onthe basis of the fluorescent signal generated by the respective labelsby standard photodetection systems, e.g. employing a system of band passfilters and photomultiplier tubes, or the like, as exemplified by thesystems described in U.S. Pat. Nos. 4,230,558; 4,811,218, or the like,or in Wheeless et al., pgs. 21-76, in Flow Cytometry: Instrumentationand Data Analysis (Academic Press, New York, 1985).

“Substantially identical” in reference to proteins or amino acidsequences of proteins in a family of related proteins that are beingcompared means either that one protein has an amino acid sequence thatis at least fifty percent identical to the other protein or that oneprotein is an isoform or splice variant of the same gene as the otherprotein. In one aspect, substantially identical means one protein, oramino acid sequence thereof, is at least eighty percent identical to theother protein, or amino acid sequence thereof.

“VEGF receptor” or “VEGFR” as used herein refers to a cellular receptorfor vascular endothelial growth factor (VEGF), ordinarily a cell-surfacereceptor found on vascular endothelial cells, as well as variantsthereof which retain the ability to bind human VEGF. VEGF receptorsinclude VEGFR1 (also known as Flt1), VEGFR2 (also know as Flk1 or KDR),and VEGFR3 (also known as Flt4). These receptors are described inDeVries et al., Science 255:989 (1992); Shibuya et al., Oncogene 5:519(1990); Matthews et al., Proc. Nat. Acad. Sci. 88:9026 (1991); Terman etal., Oncogene 6:1677 (1991); Terman et al., Biochem. Biophys. Res.Commun. 187:1579 (1992). Dimers of VEGF receptors are described inShibuya, Cell Structure and Function, 26: 25-35 (2001); and Ferrara etal., Nature Medicine, 9: 669-676 (2003). In one aspect, the inventionincludes assessing aberrant angiogenesis, or diseases characterized byaberrant angiogenesis, by measuring one or more dimers of the followinggroup: VEGFR1 homodimers, VEGFR2 homodimers, VEGFR1-VEGFR2 heterodimers,and VEGFR2-VEGFR3 heterodimers.

DETAILED DESCRIPTION OF THE INVENTION

Angiogenesis is the development of new blood vessels from existingmicrovessels. The process of generating new blood vessels plays animportant role in embryonic development, in the inflammatory response,in the development of metastases (tumor induced angiogenesis or TIA), indiabetic retinopathy, in the formation of the arthritic panus and inpsoriasis. Under normal physiological conditions, humans or animals onlyundergo angiogenesis in very specific, restricted situations. Forexample, angiogenesis is normally observed in wound healing, in fetaland embryonal development and in the formation of the corpus luteum,endometrium and placenta. The control of angiogenesis is a highlyregulated system involving angiogenic stimulators and inhibitors. Thecontrol of angiogenesis has been found to be altered in certain diseasestates and, in many cases, the pathological damage associated with thedisease is related to the uncontrolled angiogenesis.

It is well established that angiogenesis is an important requirement forthe growth and metastasis of tumors (Folkman (1987) Science 235:442-447;Folkman (1990) J. Nat. Can. Inst. 82:4-6; Talks et al. (2000) Brit. J.Haematol. 109:477-489; and Napoleone et al. (1999) Kidney Internatl.56:794-814). FIG. 1 illustrates the role angiogenesis plays in thegrowth and metastasis of a small localized tumor.

A wide variety of methods and compositions have been developed forinhibiting undesirable, aberrant angiogenesis, either by competitivelyinhibiting an angiogenesis factor or by some other mechanisms. Completeor partial suppression of vascular growth by a number of differentstrategies has been consistently associated with suppression of tumorexpansion and even reduction of tumor burden. However, none of thetreatment is effective for all the cancer patients who have the samecancer and symptoms. The traditional diagnosis is based primarily ontumor size, appearance and staging of the disease. Others have used asingle biomarker of tumor, such as expression levels of the Her-2/neugene or serum levels of VEGF, to predict patients' response totreatment, but most of the single marker alone does not correlate withit very well.

The inventors believe that since angiogenesis is a complex biologicalprocess with various factors involved, a more sophisticated analysis ofmultiple cellular components is needed to sensitively and accuratelyassess tumor angiogenesis in response to various antiangiogenicinterventions. Due to differences in pharmacogenomic profiles, patientscan have different degrees of response to the treatment of a specificantiangiogenic agent that targets a particular angiogenic factor orpathway. Further, blockage of one angiogenic pathway may triggeractivation of alternatives ones, resulting in the development ofresistance to the antiangiogenic agent. Thus, the inventors believe thatunconventional approaches should be taken to circumvent the problemsassociated with multiple, complex angiogenic pathways in the diagnosis,prevention and therapeutic treatment of diseases or conditionsassociated with undesirable, aberrant angiogenesis.

The present invention provides an innovative approach to assessingangiogenesis through sensitive, direct detection of activation of cellsthat contribute to angiogenesis, e.g., endothelial cells or tumor cells,at a molecular level. In general, activation of such cells, e.g.,endothelial cells, is detected by measuring the levels of multiplecellular components (not all interactions are cellular) and theirprotein complexes participating in a specific angiogenesis signalingpathway in endothelial cells. Compared with the currently availabletechniques for assessing angiogenesis, the methods provided in thepresent invention can sensitively and conveniently measure angiogenesisand detect the earliest indication of angiogenesis without countingblood vessels and measuring interstitial fluid pressure. Because theinventive methods can be employed to directly monitor a specificangiogenic signaling pathway, a more rational, patient-tailored therapycan be developed by targeting the specific pathway. Moreover, theinventive methods can also be used for assessing status of diseasesassociated with aberrant angiogenesis, such as the likelihood ofdeveloping the disease, presence or absence of the disease, prognosis ofthe disease and the likelihood of response or resistance to a particularanti-angiogenic therapy.

According to the invention, status of endothelial cell activation can beassessed by analyzing a patient's peripheral blood samples. Compared tobiopsy and measurement of interstitial fluid pressure, this approach ismuch less invasive and the analysis can be performed more frequently toallow closer monitoring of the patient's prognosis.

In one aspect of the invention, a method is provided for detectingactivation of endothelial cells by measuring the level of a proteincomplex (should be more than one complex at a time) in the endothelialcells in a test sample (e.g., a sample containing CEC, CECP orendothelium). The protein complex is formed between a first cellularcomponent and a second cellular component that are cellular componentsin an angiogenesis signaling pathway. The first and second cellularcomponents are preferably angiogenic receptor (e.g., receptor tyrosinekinases (RTK)), their post-translational modifications and downstreameffector proteins. The method may optionally include measurement oflevels of individual cellular components.

RTK are glycoproteins that are activated by binding of their cognateligands to the extracellular region. (Lowes V L, et al. (2002)Neurosignals 11:5-19). Ligand binding stabilizes a dimeric configurationof the extracellular domains that is required for a subsequenttransduction of the extracellular signal to the cytoplasm. This isachieved by phosphorylation of tyrosine residues on the cytoplasmicportion of the receptors themselves (trans-autophosphorylation) and ondownstream signaling proteins. Downregulation of RTK occurs viareceptor-mediated endocytosis, ubiquitin-directed proteolysis anddephosphorylation by protein tyrosine phosphatases.

As illustrated in FIG. 2, RTK comprises many families of receptors forthe epidermal growth factor (EGFR), vascular endothelial growth factor(VEGFR), angiopoietin, nerve growth factor (NGFR), fibroblast growthfactor (FGFR), platelet-derived growth factor (PDGFR), insulin andephrin receptor families, Met and Ror families. Lowes V L, et al. (2002)Neurosignals 11:5-19. In particular, VEGFR2 is the major mediator of themitogenic, angiogenic and permeability-enhancing effects of VEGF. Asillustrated in FIG. 3, in response to VEGF, endothelial cells areactivated through homodimerization of VEGFR2 which triggers a series ofdownstream signaling events involving effector proteins such as Erk (MAPkinase), Jnk (Jun kinase), MEK (MAP kinase kinase), FAK (focal adhesionkinase), p38, Src (a tyrosine kinase), PKC (phosphokinase C), Rsk(Ribosomal S6 Protein Kinase), BAD, PI3K (phosphoinositide 3-kinase),Akt (a serine-threonine protein kinase) and eNOS (endothelial nitricoxide synthase).

Examples of the protein complexes to be evaluated include, but are notlimited to, protein complexes formed by VEGFR, PDGFR, FGFR, Tie, EphAand B. The triggered intracellular protein complexes in response tothese receptors to be evaluated include, but are not limited to, Stat,BAD, RSK, P13K, FAK, Src, P70S6K, SHC, SHC, Akt, Erk, JNK, P38, and MEK.The extracellular protein interactions to be evaluated involve, but arenot limited to, VE-cadherin, heparin sulphate. In particular, thefollowing dimers are preferably evaluated: 1) VEGFR-1, 2, 3 homodimers;2) VEGFR-1, 2, 3 phosphorylation; 3) VEGFR-2/Nrp-1, Nrp-2 heterodimer;4) VEGFR-2/heparin sulphate complex; 5) VEGFR-2, VE-cadherin complex; 6)Tie 1/Tie 2 heterodimer; 7) Tie 1 endodomain/Tie 2 heterodimer; 8)VEGF/P1GF heterodimer; 9) PDGFR homo- and heterodimers; 10) EphA-1, 2,3, 4, 5, 6, 7, 8, 10, EphB-1, 2, 3, 4, 6 homo- and heterodimers; 11)Flt3 homo- and heterodimers 12) FGFR-1, 2, 3, 4 homo- and heterodimers.

In another aspect of the invention, a method is provided for detectingactivation of endothelial cells by measuring the levels of a firstcellular component and a second cellular component that participate inan angiogenesis signaling pathway in the endothelial cells in a testsample (e.g., a sample containing CEC, CECP or endothelium). The firstand second cellular components are preferably angiogenic receptor (e.g.,receptor tyrosine kinases (RTK)), their post-translational modificationsand downstream effector proteins.

By measuring levels of multiple cellular components and their complexesthat participate in a specific angiogenic pathway in endothelial cells,angiogenesis can be assessed at molecular levels to provide extremelyvaluable information on the mechanisms of action. Such information canbe used to assess status of a disease associated with undesirableangiogenesis, to select patients for clinical trials of antiangiogenicdrugs, and to guide diagnosis and therapy of such a disease by targetingthe specific angiogenic pathway.

Thus, in another aspect of the invention, a method is provided fordetermining a disease status of an individual who has or likely has adisease associated with undesirable angiogenesis. The method comprises:providing a test sample comprising endothelial cells from theindividual; measuring the level of a protein complex in the endothelialcells from the test sample, wherein the protein complex is formedbetween a first cellular component and a second cellular component thatare cellular components in an angiogenesis signaling pathway, andcomparing the level of the protein complex with a reference level of theprotein complex, wherein a difference in the level of the proteincomplex indicates the disease status of the patient. For example, a testsample containing CEC or CECP isolated from peripheral blood of theindividual can be analyzed for activation of endothelial cells which maybe indicated by increased levels of VEGFR2 homodimers and heterodimers,and optionally increased levels of phosphorylated VEGFR. As a surrogatemarker of angiogenesis, activation of endothelial cells in theindividual indicates that he/she is likely to have a disease associatedwith undesirable angiogenesis.

In yet another aspect of the invention, a method is provided forscreening patients to determine the likelihood that a patient willrespond to treatment by an anti-angiogenic agent. The method comprises:providing a test sample comprising endothelial cells from the patient tobe screened; measuring the level of a protein complex in the endothelialcells from the test sample, wherein the protein complex is formedbetween a first cellular component and a second cellular component thatare cellular components in an angiogenesis signaling pathway; andclassifying those patients having an increased level of the proteincomplex relative to a reference level characteristic of normalendothelial cells as being more likely to respond to treatment by ananti-angiogenic agent. For example, a test sample containing CEC or CECPisolated from peripheral blood of the patient (or a test samplecontaining tumor endothelium of the patient) can be analyzed foractivation of endothelial cells which may be indicated by increasedlevels of VEGFR2 homodimers and heterodimers, and optionally increasedlevels of phosphorylated VEGFR. As a surrogate marker of angiogenesis,activation of endothelial cells in the patient indicates that thepatient is likely to respond to treatment of an anti-angiogenic agent.

In yet another aspect of the invention, a method is provided fordetermining whether a patient will respond to treatment by ananti-angiogenic agent. The method comprises: providing a test samplecomprising endothelial cells from the patient who has been treated withan anti-angiogenic agent; measuring the level of a protein complex inthe endothelial cells from the test sample, wherein the protein complexis formed between a first cellular component and a second cellularcomponent that are cellular components in an angiogenesis signalingpathway; and comparing the level of the protein complex relative to areference level of the protein complex in endothelial cells in thepatient prior to the treatment, wherein a decreased level of the proteincomplex in the vascular (or CEC/CECP) endothelial cells from the testsample indicates that the patient is likely to respond to the treatmentby the anti-angiogenic agent. For example, a test sample containing CECor CECP isolated from peripheral blood of the patient (or a test samplecontaining tumor endothelium of the patient) can be analyzed forinhibition of activation of endothelial cells which may be indicated bydecreased levels of VEGFR2 homodimers and heterodimers, and optionallydecreased levels of phosphorylated VEGFR. As a surrogate marker ofangiogenesis, inhibition of activation of endothelial cells in thepatient indicates that the patient has likely responded to treatment ofan anti-angiogenic agent.

In yet another aspect of the invention, a method is provided fordetermining whether a patient has developed resistance to treatment ofan anti-angiogenic agent. The method comprises: providing a test samplecomprising endothelial cells from the patient who has been treated withan anti-angiogenic agent; measuring level of a protein complex in theendothelial cells from the test sample, wherein the protein complex isformed between a first cellular component and a second cellularcomponent that are cellular components in an angiogenesis signalingpathway; and comparing the level of the protein complex relative to areference level of the protein complex in the endothelial cells in thepatient prior to treatment, wherein a increased level of the proteincomplex in the endothelial cells from the test sample indicates that thepatient has likely developed resistance to the treatment of theanti-angiogenic agent. For example, a test sample containing CEC or CECPisolated from peripheral blood of the patient (or a test samplecontaining tumor endothelium of the patient) can be analyzed foractivation of endothelial cells which may be indicated by decreasedlevels of VEGFR2 homodimers and heterodimers, and optionally decreasedlevels of phosphorylated VEGFR. As a surrogate marker of angiogenesis,activation of endothelial cells in the patient indicates that thepatient has likely developed resistance to treatment of ananti-angiogenic agent.

In yet another aspect of the invention, a method is provided fordetecting activation of endothelial cells. The method comprises:providing a test sample comprising endothelial cells; measuring in thetest sample levels of two or more different cellular components thatparticipate in one or more angiogenesis signaling pathways, andcomparing the levels of the two or more different cellular componentswith reference levels of the two or more different cellular components,wherein a difference in the levels of the two or more different cellularcomponents indicates the disease status of the patient. Cellularcomponents that participate in one or more angiogenesis signalingpathways include, but are not limited to, surface adhesion molecules,procoagulant factors, endothelins, growth factor receptors, nitricoxide, endothelial nitric oxide synthase (eNOS), inducible nitric oxidesynthase (iNOS), prostaglandins 12 (PGI2), tissue factors, hemeoxygenase (HO), such as HO-1, tissue plasminogen activator (tPA),mitochondria superoxide dismutase (MnSOD), Cu/Zn superoxide dismutase(Cu/Zn SOD), tumor growth factor-beta (TGF-β), cyclooxygenase 1 (COX-1),cyclooxygenase-2 (COX-2), vascular cell adhesion molecule (VCAM), suchas VCAM-1, intercellular adhesion molecule (ICAM), such as ICAM-1,vascular endothelial growth factor (VEGF), VEGF receptor, E-selectin,and P-selectin. For example, indication of activation of endothelialcells can include one or more of the following: production ofendothelin, decrease in nitric oxide (NO), decrease in tissueplasminogen activator, thrombomodulin, PGI2, release of von Willebrandfactor, increase in VCAM-1, E-selectin, and plasminogen activatorinhibitor-1. More examples of such angiogenic cellular components aredescribed in detail in the following section entitled “Examples ofCellular Components and Protein Complexes in Angiogenesis SignalingPathways.” Preferably, the levels of multiple cellular components areefficiently measured by assays using releasable molecular tags.

According to the present invention, angiogenesis of an organism,preferably a human, can be assessed by directly measuring activation ofendothelial cells. The status of activation of endothelial cells can bedetermined by measuring multiple cellular components, and/or one or moreprotein complex in a sample containing endothelial cells. The status ofactivation of endothelial cells can be determined at a given time point,and correlated with future events, e.g., thereby predicting thelikelihood of developing a disease associated with undesirableangiogenesis, presence or absence of the disease, prognosis of thedisease and the likelihood of response or resistance to a particulartherapeutic regimen. The term “therapeutic regimen”, as used herein,refers to treatments aimed at the elimination or amelioration ofsymptoms and events associated with conditions of a disease, inparticular, a disease associated with undesirable angiogenesis. Inaddition to pharmaceutical interventions, such treatments can includewithout limitation one or more of alteration in diet, lifestyle, andexercise regimen; invasive and noninvasive surgical techniques; andradiotherapy. Pharmaceutical interventions can include administration ofa therapeutic agent, such as an antiangiogenic agent. Specific examplesof such an antiangiogenic agent are described in detail in the followingsection entitled “Examples of Antiangiogenic Agents.”

Any of the above methods may further comprise the step of treating theindividual or patient with a pharmaceutically effective amount of atherapeutic agent, preferably an anti-angiogenic agent. Thepharmaceutically effective amount of a therapeutic agent depends onseveral factors such as, the age, weight, and the severity of thecondition under treatment, as well as the route of administration,dosage form and regimen and the desired result, and additionally thepotency of the particular therapeutic agent employed in the composition.In addition, account should be taken of the recommended maximum dailydosages for the therapeutic agent. A unit dosage formulation such as atablet or capsule, will usually contain, for example, from 0.1 mg to 500mg of an antiangiogenic agent. Preferably, a unit dose formulation willcontain 0.1 to 100 mg of an antiangiogenic agent. The antiangiogenicagent may be administered to the patient up to six times daily,conveniently 1 to 4 times daily and preferably 1 to 2 times daily, sothat a dose of the antiangiogenic agent in the general range of 0.01 to100 mg/kg, preferably 0.1 to 10 mg/kg, more preferably 0.1 to 5 mg/kg,is administered daily. A therapeutically effective amount of thetherapeutic agent for treating the disease can be administered prior to,concurrently with, or after the onset of the disease or symptom.

The following is a detailed description of various aspects of theinvention, including preferred embodiments, as well as examples ofvarious elements of the inventions.

1. Examples of Cellular Components and Protein Complexes in AngiogenesisSignaling Pathways

The protein complexes or protein-protein interactions play veryimportant structural and functional roles in almost all biologicalprocesses. For example, cell surface receptor-mediated signaltransduction, nuclear receptor-mediated signaling events, transcription,translation, post-translational modification, and protein secretion, allrequire protein-protein interaction, either transient, or stableinteraction. Protein interaction can be receptor-receptor interaction,receptor-ligand interaction, receptor-adaptor protein interaction,non-receptor protein kinase-kinase interaction, or kinase-adaptorprotein interaction. Protein complexes referred here can be those formedin different cell types or tissues, such as endothelial cells andpericytes, which triggers the diverse array of molecular interaction andsignaling leading to angiogenesis.

Several families of receptor tyrosine kinases (RTKs) have emerged ascritical mediators of angiogenesis: vascular endothelial growth factor(VEGF), fibroblast growth factor (FGF), PDGF (platelet-derived growthfactor), Tie, and Ehp RTK families (Gale, et al. Genes Dev. 199913:1055-1066; Yancopoulos, et al. Nature 2000 407:242-248). They arepotent angiogenic factors, functioning via their cognate receptors,during embryogenesis and tumorogenesis.

Vascular endothelial growth factor is a potent, multifunctional,endothelial cell specific growth factor. It stimulates proliferation andmigration of endothelial cells and is regarded as a key contributor tothe growth of cancer and vascular diseases. VEGF activities are mediatedby high affinity receptor tyrosine kinases VEGFR1 (Flt-1) and VEGFR2(KDR or Flk-1), and they are mainly expressed on endothelial cells (F),as well as, for example, on stroma cells or tumor cells, see e.g., U.S.Pat. Pub. No. 2004/0229293, filed Mar. 30, 2004, the contents of whichis incorporated by reference in its entirety; Neuchrist et al., 2001,Laryngoscope, 111(10):1834-41, the contents of which is incorporated byreference in its entirety. Recent studies have found a third VEGFreceptor, neuropilin (NRP-1), expressed by endothelial cells and tumorcells (Soker, et al. Cell 1998 92:735-745). Induction of NRP-1expression in tumor cells in vivo resulted in larger and more vasculartumors (Soker, et al. J. Biol. Chem 1996 271:5761-5767; Miao, et al.FASEB J. 2000 14:2532-2539).

The receptor tyrosine kinases Tie-1 and Tie-2, predominantly expressedon endothelial cells, have also been shown to be essential fordevelopmental vascularization where they promote microvessel maturationand stability (Tallquist, et al. Oncogene, 1999 18:7917-7932). Severalligands, designated the angiopoietins, have been identified for Tie-2(Davis, et al. Cell 1996 87:1161-1169; Kim, et al. FEBS Lett, 1999443:353-356; Maisonpierre, et al. Science 1997 277: 55-60; Mezquita, etal. Biochem. Biophys Res Commun 1999 260:492-298; Valenzuela, et al.Proc. Natl, Acad, Sci. USA 1999 96:1904-1909). Angiopoietin-1 (Ang-1)(Tallquist, et al. Oncogene, 1999 18:7917-7932) and angiopoietin-2(Ang-2) are the best characterized of the ligands. Binding of Ang-1induces tyrosine phosphorylation of Tie-2 and activation of itssignaling pathway, whereas Ang-2 has been reported to antagonize theseeffects in endothelia cells (Davis, et al. Cell 1996 87:1161-1169;Maisonpierre, et al. Science 1997 277: 55-60).

The Eph family of RTKs and their ligands (EphB and EphA) are alsoritical regulators for vascular development. For example, the expressionof ephrinA1 and its receptor EphA2 was detected in breast cancers andassociated vasculature (Ogawa, et al. Oncogene 2000 19:6043-6052).Moreover, blocking EphA receptor activation impaired tumor angiogenesis(Brantley, et al. Oncogene 2003 21:7011-7026). Chang, et al. hadprovided evidence that Eph RTKs and their ligands are necessary forinduction of maximal angiogenesis by VEGF. Furthermore it was revealedthat ephrinA1 is a downstream target gene product that is induced byVEGF (Cheng, et al. Mol. Cancer Res 2002 1:2-11).

Fibroblast growth factors (FGFs) form a large family of structurallyrelated, multifunctional proteins that regulates an array of biologicalprocesses. Like VEGF, stimulates survival, proliferation, migration anddifferentiation of primary and stable endothelial cells. Dimeric FGF-2binds to two FGF receptors in the presence of heparin or heparinsulfate, which leads to receptor dimerization and intermolecularautophosphorylation (DiGabriele, et al. Nature 1998 393:812-817).

PDGFs and their cognate tyrosine kinase alpha- and beta-receptors alsoinvolved in multiple tumor-associated processes including autocrinegrowth stimulation of tumor cells, recruitment and regulation of tumorfibroblasts, and stimulation of tumor angiogenesis. PDGF receptorsignaling has been implicated in tumor pericyte recruitment. Theinteraction between endothelial cells and pericytes plays a criticalrole in stabilizing blood vessels (particularly the newly formedvessels), endothelial cell proliferation and survival (Erber, et al.FASEB J. 2004 18:338-340). PDGF receptors in the tumor stroma have alsobeen shown to function as regulators of tumor interstitial fluidpressure (Pietra, et al. Cancer Res. 2001 61:2929-2934).

It is well established that the binding of these ligand typicallyresults their receptor dimerization and initiates the downstream signaltransduction event, mainly through the phosphorylation of specifictyrosine residues within the intracellular domain of the receptor.

Many intracellular signaling proteins are activated directly throughreceptor binding, such as Crk and PLC-r (phospholipase C-r), in the caseof FGFR-mediated signaling, to activate MAPK (mitogen-activated proteinkinase), JNK kinases and to mediate Ca2++ release and DAG generation,respectively. Some direct protein-protein interactions have also beenidentified in VEGFR-1-mediated signaling. For example, tyrosine residues1213 and 1333 of VEGFR-1 have been shown to interact with severalsignaling molecules, based on their SH2-domain-binding specificity. InVEGFR-2, Sck (She-like protein), PLC-r and VRAP (VEGFreceptor-associated protein) directly interact with the receptor (Cross,et al. 2001 Trends in Pharmacological Sci. 22:201-207).

Some proteins might interact with the receptor via indirect mechanisms,or the direct binding (or binding sites) is yet to be identified. InFGFR triggered signaling, Crk might transduce its signal through thecomplex of FRS-2, Shp-1, Grb2 and Sos before it relayed to Ras, Raf, MEKand MAPK pathway. Grb2 has been postulated to interact with Shc alongwith Sos, which would ultimately modulate MAPK pathway (Cross, et al.2001 Trends in Pharmacological Sci. 22:201-207). Shc and Grb14 have beenimplicated in FGFR-1 activation by cascading signaling to p42/44 MAPKleading to proliferation. PI3K might also participate in FGF signalingas a downstream molecule of PLC-r. In VEGFR-2, many intracellularsignaling proteins, such as Akt, STATs (signal transducers andactivators of transcription), FAK (focal adhesion kinase), p38 MAPK,eNOS (endothelia nitric oxide synthase), Src and PI3K (phosphoinositide3-kinase) are implicated as important downstream signaling molecules inresponse to activation of VEGFR-2, even though the exact mechanism(s) ofthe protein complex formation remains to be elucidated (Cross, et al.2001 Trends in Pharmacological Sci. 22:201-207).

Akt, a serine/threonine kinase, functions intracellularly to integrateupstream signals from receptors (e.g. VEGFRs, HER2/neu, IGFR), andregulate the phosphorylation of its multiple downstream effectors, suchas NFkB, mTOR, Forkhead, BAD, GSK-3 and MDM-2. These phosphorylationevents in turn can also modulate the effects of Akt on cell growth,proliferation, protection from pro-apoptotic stimuli, and stimulation ofangiogenesis (Mitsiades, et al. Curr Cancer Drug Targets, 20044:235-256).

STAT is another critical pathway downstream of VEGFRs. It has been shownthat dominant-negative STAT3 completely abolished VEGF-induced nucleartranslocation of phosphorylated STAT3 and inhibited endothelialmigration in vitro (Yahata, et al. J. Biolo. Chem. 2003278:40026-40031), which demonstrated the role of phosphorylated STAT3 inVEGF-triggered signaling pathways. Most recent studies have provided invivo evidence, which indicated that the expression of phosphorylatedSTAT3 and STATS, but not phosphorylated STAT1 or STAT6, were observed tobe elevated only in ovarian epithelial carcinoma. Normal ovariantissues, or benign ovarian tumors expressed significantly lower levelsof phosphorylated STAT3 and STATS. The over expression of phosphorylatedSTAT3 and STATS appeared to correlate with the activation of VEGFR(Chen, et al. Gynecol. Oncol. 2004 94:630-635).

The essential roles of protein-protein interaction extend beyond thecell surface receptor-ligand interaction and its tirggered intracellularpathways. Though the key receptors, their ligands (e.g. growth factors)and the related networks formed by the intracellular kinases andproteins play the central roles in signal transduction leading topro-angiogenesis, the extracellular matrix components and othermolecules are also indispensable part of the angiogenesis processes invivo. Indeed, several extracellular molecules have been shown to possessunique anti-angiogenic function (Fotsis, et al. Nature 368:237-239;Polakowski, et al. Am J. Pathol. 1993 143-507-517). The mechanisms ofaction of most of these molecules are poorly defined, nonetheless, someof these angiogenesis inhibitors were found to be either heparin analogsor heparin binding substances (Taylor, et al. Nature, 1982 297:307-312;Jouan, et al. Blood 1999 94:984-993). Heparins are linear anionicpolysaccharide chains, and they are typically heterogeneously sulphatedon alternating L-iduronic and D-glusocamino sugars. They are nearlyubiquitous in animal tissues as heparin sulphate proteoglycans on cellsurfaces and in the extracellular matrix (DiGabriele, et al. Nature 1998393:812-817).

Heparin sulphate can bind to the various forms of VEGFAs, for example,to modulate the affinity of VEGFAs to its receptor. In fact the bindingof VEGF165 to its receptors requires cell surface heparin sulfates andthe binding can be regulated by exogenous heparin (Gitay-Goren, et al.J. Biol. Chem. 1992 267:6093-6098). These observations together with therecent results showing that heparin-degrading enzymes can inhibit thetumor angiogenesis (Sasisekharan, et al. Proc. Natl. Acad. Sci. USA,1994 91, 1524-1528) suggest that heparin sulfates may play an importantregulatory role in the angiogenesis processes. The requirement forheparin-like molecule to modulate receptor-ligand binding is not uniquefor VEGFAJVEGFR interaction, as it has been well-studied that FGFbinding to FGFR and the activation of the receptor also requiresheparin-like molecules (Spivak-Kroizman, et al. Cell 1994 79:1015-1024).

In some embodiments of the present invention, the cellular componentsparticipating in angiogenesis pathways include receptors for angiogenicgrowth factors. As described above, these receptors belong to the familyof the receptor tyrosine kinase and are intimately involved in tumordevelopment and metastasis. Example of the receptor tyrosine kinaseinclude, but are not limited to, receptor for fibrin (VE-cadherin),receptors for VEGF (Flt1 and KDR/flk-1), receptor for VEGF-C and VEGF-D(Flt4), receptor for VEGF-165 (NP-1 and NP-2), receptors forangiopoeitin-1, -2, -3, and -4 (Tie1 and Tie 2), receptors for FGF(FGF-R1, -R2, -R3 and -R4), receptor for PDGF (PDGF-R), receptor forephrine A1-5 (Eph A1-8), and receptor for ephrine B1-5 (Eph B1-8).

The cellular components participating in angiogenesis pathways alsoinclude receptors for anti-angiogenic protein factors. Examples of suchreceptors include, but are not limited to, receptor for angiostatin(angiostatin-R, also called Annexin II), receptor for angiostadin(angiostadin binding protein I), low-affinity receptors for glypicans,receptor for endostatin (endostatin-R), the receptor for endothelin-1(endothelin-A receptor), receptor for angiocidin (angiocidin-R), thereceptor angiogenin (angiogenin-R), receptors for thromospondin-1 andthromospondin-2 (CD36 and CD47), and the receptor for tumstatin(tumstatin-R).

The cellular components participating in angiogenesis pathways alsoinclude G protein coupled receptors (GPCR). Examples of such GPCRinclude, but are not limited to, receptor for sphingosie-1-phosphate orSPP and for lysophosphatidic acid or LSA (edg receptor), cytokinereceptors such as receptor for tumor necrosis factor-α or TNF-α (TNF-αreceptor) and receptor for interleukin-8 or IL-8 (IL-8 receptor),protease receptors such as receptor for urokinase (urokinase receptor),and integrins such as receptor for thromospondin-1 and -2 (αvβ3 integrinand α2vβ1 integrin) and receptor for fibronectin (αvβ3 integrin), andmatrix metalloproteases.

Various protein complexes formed by the cellular components inendothelial cells play important roles in activation of endothelialcells, leading to angiogenesis. For example, in response to the releaseof VEGF by the tumor mass, angiogenesis is stimulated in adjacentendothelial cells via formation of protein complexes of receptors forVEGF. Normally, capillary endothelial cells turn over extremely slowly(thousands of days) and are kept quiescent through contact withspecialized cells, called pericytes. When VEGF is expressed by the tumormass, endothelial cells closely adjacent to the VEGF+tumor cells willup-regulate expression of VEGF receptor (VEGFR) molecules KDR/flk-1and/or flt-1. Upon binding of their ligand, these receptors dimerize andtransduce an intracellular signal through tyrosine phosphorylation.Binding of VEGF to its cognate VEGF receptors on endothelial cellsinitiates a chemotaxic effect in these cells through signaltransduction. New capillaries sprout from the endothelial vesselstowards the VEGF+ tumor cells. During angiogenesis, the endothelialcells rapidly proliferate by dividing up to every five days. Thus, bymeasuring formation of protein complexes of VEGFR and/or other cellularcomponents in the VEGF-dependent angiogenic signaling pathway,activation of endothelial cells can be detected in a pathway-specificmanner.

The Tie receptors for angiopoietin also play important roles instimulation of angiogenesis. The Tie receptors, Tie-1 and Tie-2 (Tiestands for Tyrosine kinase receptors with immunoglobulin and EGFhomology domains), are among many receptor tyrosine kinases (RTK)expressed on endothelial cells. Mustonen (1995) J. Cell. Biol. 129:895).Angiopoietin-1 (Ang-1) is the major physiological ligand for Tie-2 whichis responsible for recruiting and sustaining periendothelial supportcells (Davis et al. (1996) Cell 87:1161-1169). Angiopoietin-2 (Ang-2) isfound to be responsible in disrupting vessel formation in the developingembryo by antagonizing the effects of Ang-1 and Tie 2. Tie 1 and Tie 2are homologous to each other, but unlike the VEGF receptors, theycontain matrix association motifs in their extracellular domains. Bothare expressed very early in development (Dumont et al. (1995) Dev. Dyn.203:80-92). Tie-2 is expressed in the blood islands and inintraembryonic angioblasts, where it appears earlier than von Willebrandfactor. The Tie 1 and Tie 2 receptors are single-transmembrane, tyrosinekinase receptors. They are the only receptor tyrosine kinases, otherthan those receptors for VEGF, that are largely restricted toendothelial cells in their expression.

The Tie receptors are proteins of approximately 125 kDa, with a singleputative transmembrane region. The extracellular domain of thesereceptors is uniquely divided into three regions that have a pattern ofcysteine expression found in EGF-like domains; two regions that havesome weak homology to and structural characteristics ofimmunoglobulin-like domains; and three regions with homology to thefibronectin III repeat structure. The intracellular portion of Tie 2 ismost closely related (-40% identity) to the kinase domains of FGF-R1,PDGF-R and c-kit. The intracellular portions of Tie 2 contain all of thefeatures of tyrosine kinases. It is well established that Tie 2 and itsligand Ang1 play crucial roles in the maturation of blood vessels inhealthy angiogenesis by inducing endothelial cells to recruit andincorporate pericytes and smooth muscle cells into the vessel wall, andremodeling and stabilizing the immature network of blood vessels formedunder the effect of VEGF. Herbst et al. (2002) Hematol. Oncol. Clin. N.Am. 16:1125-1171.

In some embodiments of the present invention, the cellular componentsand protein complexes participating in angiogenesis signaling pathwaysinclude, but are not limited to,

1) VEGFR-1, 2, 3 homodimers and heterodimers (Ferrara (2004) EndocrineReviews 25(4):581-611);

2) phosphorylated VEGFR-1, 2, 3 (Ferrara (2004), supra);

3) VEGFR-2/Nrp-1, VEGFR-2/Nrp-2 heterodimers (Ferrara (2004), supra; andHerbst et al. (2002); Neuropilin-1 forms a ternary complex with VEGFR-2and VEGF and potentiates the effects of VEGF);

4) VEGFR-2/heparin sulphate complex (Nicosia (1998) Am. J. Pathol.153:11-16; Heparin binds to certain isoforms of VEGF with a higheraffinity than other isoforms and sequesters VEGF to the extracellularmatrix);

5) VEGFR-2NE-cadherin complex;

6) Tie 1/Tie 2 heterodimer (Brindle et al. (2000) J. Biol. Chem. 275:39741-39746);

7) Tie 1 endodomain/Tie 2 heterodimer (Brindle et al. (2000), supra);

8) VEGF/P1GF heterodimer (Ferrara (2004), supra);

9) PDGFR homo and heterodimers (Hanahan et al. (2003) J. Clin. Invest.111:1287-1295; PDGFR signaling is critical for pericyte-endothelialassociation (vessel maturation) in tumors);

10) EphA-1, 2, 3, 4, 5, 6, 7, 8, 10, EphB-1, 2, 3, 4, 6 homo andheterodimers (Chen et al. (2002) Mol. Cancer Res. 1:2-11; and Herbst etal. (2002), supra; Ephrins must be tethered to membranes to activatetheir receptors (Ephs); Ephrins are involved in adult and developmentalsprouting angiogenesis, juxtacrine cell-to-cell contacts, adhesion toextracellular matrix, and migration; and Eph receptors are overexpressedin many types of tumor cells and tumor-associated endothelial cell);

11) FLT3 dimers;

12) FGFR-1, 2, 3, 4 homo and heterodimers (Claesson-Welsh et al. (2001)TRENDS in Pharm Sci, Vol. 22 No. 4: 201-207; Herbst et al. (2002),supra; FGFs are potent angiogenic molecules and stimulate endothelialcell mitosis, migration, morphogenesis, and survival);

13) Stat3 complexes and phosphorylated Stat3

14) BAD complexes and phosphorylated BAD (Zachary (2003) BiochemicalSociety Transactions 31:1171-1177);

15) Rsk complexes and phosphorylated Rsk;

16) PI3K complexes and phosphorylated PI13K (Rahimi et al. (2001) J.Biol. Chem. 276:17686-17692);

17) Fak complexes and phosphorylated Fak (Claesson-Welsh et al. (2004).J. Biol. Chem. March 16, 2004);

18) Src complexes and phosphorylated Src (Fujita et al. (2002) BMCBiochemistry 3:32);

19) P70S6K complexes and phosphorylated P70S6K;

20) SHC complexes and phosphorylation (Park et al. (2004) Proc. Natl.Acad. Sci. 101:2345-2350);

21) Stat1 complexes and phosphorylated Stat 1;

22) Stat5 complexes and phosphorylated Stat5;

23) Akt complexes and phosphorylated Akt (Ferrara (2004), supra);

24) Erk complexes and phosphorylated Erk (Ferrara (2004), supra);

25) Jnk complexes and phosphorylated Jnk (Zachary (2003), supra);

26) P38 complexes and phosphorylated P38 (Zachary (2003), supra);

27) MEK complexes and phosphorylated MEK (Ferrara (2004), supra);

28) PLC-γ complexes and phosphorylated PLC-γ (Shibuya et al. (2001). “Asingle autophosphorylation site on KDR/Flk-1 is essential forVEGF-A-dependent activation of PLC-γ and DNA synthesis in vascularendothelial cells”);

29) Shb complexes and phosphorylated Shb (Claesson-Welsh et al. (2004),supra); and

30) Her-1, 2, 3, 4 homodimers and heterodimers.

It is noted that such cellular components or protein complexes can bedetected by the methods of the present invention on any cells thatcontribute to or promote angiogenesis, including, e.g., endothelialcells, stroma cells or tumor cells. While detection on endothelial cellsis exemplified throughout, it should be well understood by the skilledartisan that the methods of the present invention can be applied to anycells suspected of contributing to or promote angiogenesis, e.g., stromacells or tumor cells.

In addition, the cellular components participating in angiogenesissignaling pathways also include secreted angiogenic factors andintercellular adhesion molecules. Studies have revealed that activationof endothelial cells increase the expression of surface adhesionmolecules, express and release procoagulant factors, and secretevasoactive compounds including endothelins, nitric oxide, andprostaglandins. All of these substances have been implicated in thepathophysiology of a variety of diseases (Corti et al. “Endothelialdysfunction and hypertension” In: Vascular Endothelium in HumanPhysiology and Pathophysiology, edited by Vallance P. J. T. and Webb D.J. Amsterdam: Harwood, 2000, pp. 109-128; Goligorsky et al. (2001)Hypertension 37:744-874; Haller (1997) Drugs 53:1-10; Naicker et al.(2001) Pharmacol Ther. 90:61-88; Smith et al. (1993) Physiol Pharmacol.71:76-87; Vapaatalo et al. (2001) Med Sci Monit. 7:1075-1085). Specificexamples of the cellular components include surface adhesion molecules,procoagulant factors, endothelins, growth factor receptors, nitricoxide, eNOS, iNOS, PGI2, tissue factors, heme oxygenase (HO), such asHO-1, tPA, MnSOD, Cu/Zn SOD, TGF-β, COX-1, COX-2, VCAM, such as VCAM-1,ICAM, such as ICAM-1, VEGF, E-selectin, and P-selectin.

2. Preparation of Samples

Samples containing cellular components of endothelial cells may comefrom a wide variety of sources for use with the present invention,including cell cultures, animal or plant tissues, patient biopsies,patient blood, bodily fluid, and the like. Preferably, samples are fromhumans. Samples are prepared for assays of the invention usingconventional techniques, which may depend on the source from which asample is taken.

1) Isolation of Circulating Endothelial Cells and CirculatingEndothelial Cell Progenitors

Circulating endothelial cells (CEC) and circulating endothelial cellprogenitors (CECP) have been used as surrogate markers of tumorangiogenesis. Mancuso et al. (2001) Blood 97:3658-3661; and Monestiroliet al. (2001) Cancer Res. 4341-4344. According to the present invention,multiple cellular components and their interactions in CEC and CECP areanalyzed to provide information for assessing angiogenesis, monitoringdisease status, predicting treatment response, etc.

CEC and CECP can be isolated from blood specimens by using varioustechniques available in the art. Blood specimens can be obtained usingmethods of sampling known in the art, such as venipuncture. Venous bloodcan be drawn into vessels, such as tubes, and optionally treated withsilicon and EDTA for subsequent analysis. As used herein, the terms“blood sample” and “blood specimen” are used interchangeably to refer toa volume of blood that is preferably, although not necessarily, removedfrom the patient's peripheral circulatory system.

The blood specimen may be obtained by isolating a volume of blood fromthe bloodstream. however, other means for sampling the patient's bloodcan be utilized, such as in-dwelling systems. For example, anin-dwelling catheter can be utilized to remove a volume of blood.

The blood specimen is treated to remove lower molecular weightcontaminants, for example, by dialysis. Spectra/Por membrane dialysistubing with a desired molecular weight cut-off (MWCO) can be utilized.Other products that can be utilized include hollow fiber concentrationsystems consisting of regenerated cellulose fibers for larger volumes; amultiple dialyzer apparatus with a sample size for one to fivemilliliters; and a multiple microdializer apparatus, convenient forsamples in plates with 96 wells, for example. Other equivalenttechniques include passage through a column holding a resin or mixtureof resins suitable for removal of low molecular weight materials. Resinssuch as BIOGEL (BIORAD, Hercules, Calif.) and SEPHAROSE (PHARMACIA,Piscataway, N.J.) and others are well-known to the skilled artisan. Thetechnique of dialysis, or equivalent techniques with the same function,are intended to remove low molecular weight contaminants from the bloodspecimen.

Specific antibodies (e.g., polyclonal or monoclonal antibodies) raisedto a particular cellular component of CEC or CECP, such as VEGFR, can beused to isolate CEC or CECP in blood specimens. The term “antibody” asused herein, includes monoclonal and polyclonal antibodies as well asantibody fragments which bind specifically but reversibly to thedescribed epitope. It is preferred that the antibody or antibodyfragment is derived from a monoclonal antibody or antibody fragment.Preparation of monoclonal and polyclonal antibodies to an antigenrepresenting an endothelial cell activation marker can be achieved usingany known method, and for example, those described in Zola, H. (1988)“Monoclonal Antibodies—A manual of techniques” CRC Press, andAntibodies: A Laboratory Manual, Harlow & Lane; Cold Spring Harbor(1988), incorporated herein by reference. Specific high affinity bindingproteins can be used in place of antibodies, and can be made accordingto methods known to those in the art. For example, proteins that bindspecific DNA sequences may be engineered (U.S. Pat. No. 5,096,815), andproteins that bind a variety of other targets, particularly proteintargets (U.S. Pat. No. 5,233,409; U.S. Pat. No. 5,403,484) can beengineered and used in the present invention. Antibodies and/or bindingproteins can be incorporated into large scale diagnostic or assayprotocols that can involve immobilization of the antibody or bindingprotein onto a surface, such as a multi-well plate assay, or on beads,for example.

General techniques to be used in performing various immunoassays areknown to those of ordinary skill in the art. General descriptions ofthese procedures are provided in manuals of the art (Ichikawa, E. etal., (1988) Enzyme Immunoassay, Igaku-shoin, Tokyo, N.Y.; Hallow, E. etal., Antibodies: A Laboratory Manual, CSH Press, N.Y.).

In a preferred embodiment, CEC and/or CECP are isolated byimmunomagnetic isolation or enrichment involving monoclonal antibodiesagainst a CEC or CECP cellular component coupled to magnetic beads.Immunomagnetic isolation or enrichment may be carried out using avariety of techniques and materials known in the art, as disclosed inthe following representative references that are incorporated byreference: Terstappen et al., U.S. Pat. No. 6,365,362; Terstappen etal., U.S. Pat. No. 5,646,001; Rohr et al., U.S. Pat. No. 5,998,224;Kausch et al., U.S. Pat. No. 5,665,582; Kresse et al., U.S. Pat.6,048,515; Kausch et al., U.S. Pat. No. 5,508,164; Miltenyi et al., U.S.Pat. No. 5,691,208; Molday, U.S. Pat. No. 4,452,773; Kronick, U.S. Pat.No. 4,375,407; Radbruch et al., chapter 23, in Methods in Cell Biology,Vol, 42 (Academic Press, New York, 1994); Uhlen et al., Advances inBiomagnetic Separation (Eaton Publishing, Natick, 1994); Safarik et al.,J. Chromatography B, 722: 33-53 (1999); Miltenyi et al., Cytometry, 11:231-238 (1990); Nakamura et al., Biotechnol. Prog., 17: 1145-1155(2001); Moreno et al., Urology, 58: 386-392 (2001); Racila et al., Proc.Natl. Acad. Sci., 95: 4589-4594 (1998); Zigeuner et al., J. Urology,169: 701-705 (2003); Ghossein et al., Seminars in Surgical Oncology, 20:304-311 (2001).

The preferred magnetic particles for use in carrying out this inventionare particles that behave as colloids. Such particles are characterizedby their sub-micron particle size, which is generally less than or about200 nanometers (nm) (0.20 microns), and their stability to gravitationalseparation from solution for extended periods of time. In addition tothe many other advantages, this size range makes them essentiallyinvisible to analytical techniques commonly applied to cell analysis.Particles within the range of 90-150 nm and having between 70-90%magnetic mass are contemplated for use in the present invention.Suitable magnetic particles are composed of a crystalline core ofsuperparamagnetic material surrounded by molecules which are bonded,e.g., physically absorbed or covalently attached, to the magnetic coreand which confer stabilizing colloidal properties. The coating materialshould preferably be applied in an amount effective to prevent nonspecific interactions between biological macromolecules found in thesample and the magnetic cores. Such biological macromolecules mayinclude sialic acid residues on the surface of non-target cells,lectins, glyproteins and other membrane components. In addition, thematerial should contain as much magnetic mass/nanoparticle as possible.The size of the magnetic crystals comprising the core is sufficientlysmall that they do not contain a complete magnetic domain. The size ofthe nanoparticles is sufficiently small such that their Brownian energyexceeds their magnetic moment. As a consequence, North Pole, South Polealignment and subsequent mutual attraction/repulsion of these colloidalmagnetic particles does not appear to occur even in moderately strongmagnetic fields, contributing to their solution stability. Finally, themagnetic particles should be separable in high magnetic gradientexternal field separators. That characteristic facilitates samplehandling and provides economic advantages over the more complicatedinternal gradient columns loaded with ferromagnetic beads or steel wool.Magnetic particles having the above-described properties can be preparedby modification of base materials described in U.S. Pat. Nos. 4,795,698,5,597,531 and 5,698,271, all of which patents are incorporated byreference.

In a particular embodiment, substantially pure CEC and CECP are isolatedby using the immunomagnetic isolation/enrichment technique described inKinzler et al. (2000) Science 289:1197-1202, which is hereinincorporated by reference. Briefly, the epithelial and hematopoieticcell fractions in the peripheral blood samples are sequentially removedvia negative selection via antibody-linked magnetic beads (BerEP4beads-Epithelial, CD45 beads-Pan leukocyte, CD64 beads-Macrophages, andCD14 beads-Monocytes, CD146 beads-endothelial cells). The remainingcells are stained with P1H12 antibodies (need to provide info on P1P12antibody) and are isolated via positive selection with magnetic beads.

2) Preparation of Samples Containing Endothelium

Also according to the present invention, multiple cellular componentsand their interactions in endothelial cells in endothelium (e.g., tumorendothelium) are analyzed to provide information for assessingangiogenesis, monitoring disease status and predicting treatmentresponse.

The endothelium is located at the interface between the blood and thevessel wall, and is composed of endothelial cells. Each endothelial cellis anchored to an underlying basal lamina; and individuals cells areanchored together by adhesion junctions. The tissue samples containingendothelium can be prepared from biopsy and medical specimen of any typeof tissue containing blood vessels, the guidance for which is providedin the following references: Bancroft J D & Stevens A, eds. Theory andPractice of Histological Techniques (Churchill Livingstone, Edinburgh,1977); Pearse, Histochemistry. Theory and applied. 4^(th) ed. (ChurchillLivingstone, Edinburgh, 1980).

Examples of tissue samples that contain endothelium include, but are notlimited to, breast, prostate, ovary, colon, lung, endometrium, stomach,salivary gland or pancreas. The tissue sample can be obtained by avariety of procedures including, but not limited to surgical excision,aspiration or biopsy. The tissue may be fresh or frozen. In oneembodiment, assays of the invention are carried out on tissue samplesthat have been fixed and embedded in paraffin or the like; therefore, insuch embodiments a step of deparaffination is carried out. A tissuesample may be fixed (i.e. preserved) by conventional methodology [Seee.g., “Manual of Histological Staining Method of the Armed ForcesInstitute of Pathology,” 3^(rd) edition (1960) Lee G. Luna, H T (ASCP)Editor, The Blakston Division McGraw-Hill Book Company, New York; TheArmed Forces Institute of Pathology Advanced Laboratory Methods inHistology and Pathology (1994) Ulreka V. Mikel, Editor, Armed ForcesInstitute of Pathology, American Registry of Pathology, Washington, D.C.One of skill in the art will appreciate that the choice of a fixative isdetermined by the purpose for which the tissue is to be histologicallystained or otherwise analyzed. One of skill in the art will alsoappreciate that the length of fixation depends upon the size of thetissue sample and the fixative used. By way of example, neutral bufferedformalin, Bouin's or paraformaldehyde, may be used to fix a tissuesample.

Generally, a tissue sample is first fixed and is then dehydrated throughan ascending series of alcohols, infiltrated and embedded with paraffinor other sectioning media so that the tissue sample may be sectioned.Alternatively, one may section the tissue and fix the sections obtained.By way of example, the tissue sample may be embedded and processed inparaffin by conventional methodology (See e.g., “Manual of HistologicalStaining Method of the Armed Forces Institute of Pathology”, supra).Examples of paraffin that may be used include, but are not limited to,Paraplast, Broloid, and Tissuemay. Once the tissue sample is embedded,the sample may be sectioned by a microtome or the like (See e.g.,“Manual of Histological Staining Method of the Armed Forces Institute ofPathology”, supra). By way of example for this procedure, sections mayhave a thickness in a range from about three microns to about twelvemicrons, and preferably, a thickness in a range of from about 5 micronsto about 10 microns. In one aspect, a section may have an area of fromabout 10 mm² to about 1 cm². Once cut, the sections may be attached toslides by several standard methods. Examples of slide adhesives include,but are not limited to, silane, gelatin, poly-L-lysine and the like. Byway of example, the paraffin embedded sections may be attached topositively charged slides and/or slides coated with poly-L-lysine.

If paraffin has been used as the embedding material, the tissue sectionsare generally deparaffinized and rehydrated to water. The tissuesections may be deparaffinized by several conventional standardmethodologies. For example, xylenes and a gradually descending series ofalcohols may be used (See e.g., “Manual of Histological Staining Methodof the Armed Forces Institute of Pathology”, supra). Alternatively,commercially available deparaffinizing non-organic agents such asHemo-De® (CMS, Houston, Tex.) may be used. For mapping tumorendothelium, the sample preparation method described in Schnitzer et al.(2004) Nature 429:629-636 may be used.

3. Assays for Measuring Cellular Components and Their Complexes

A variety of assays may be used to detect protein-protein interactionsand measure multiple proteins simultaneously.

1) Immunoaffinity-Based Methods

In one embodiment, immunoaffinity-based methods, such asimmunoprecipitation or ELISA, is used to detect complexes of cellularcomponents in endothelial cells in a test sample containing CEC, CECP orendothelium. For example, to detect the protein complex formed by VEGFR2and VE-cadherin, anti-VEGFR2 antibody can be used to immunoprecipitatecomplexes comprising VEGFR2 from CEC, and the resultingimmunoprecipitant is then probed for the presence of VE-cadherin byimmunoblotting.

In other embodiments, ELISA or antibody “sandwich”-type assays can beused to detect complexes of cellular components in endothelial cells ina test sample containing CEC, CECP or endothelium. For example,antibodies to VEGFR2 are immobilized on a solid support, contacted withCEC lysate, washed, and then exposed to antibody against VE-cadherin.Binding of the latter antibody, which may be detected directly or by asecondary antibody conjugated to a detectable label, indicates thepresence of the complex of VEGFR2 and VE-cadherin in CEC.

2) Cross-Linking Assays

Chemical or UV cross-linking may also be used to covalently joinheterodimers on the surface of living cells. Hunter et al., Biochem. J.,320:847-53. Examples of chemical cross-linkers includedithiobis(succinimidyl) propionate (DSP) and3,3′dithiobis(sulphosuccinim-idyl) propionate (DTSSP). In oneembodiment, cell extracts from chemically cross-linked endothelial cellsare analyzed by SDS-PAGE and immunoblotted with antibodies to differentcellular components. A supershifted band of the appropriate molecularweight most likely represents complex formation. This result may beconfirmed by subsequent immunoblotting.

3) Fluorescence Resonance Energy Transfer (FRET)

FRET may also be used to detect complexes of cellular components inendothelial cells in a test sample containing CEC, CECP or endothelium.FRET detects protein conformational changes and protein-proteininteractions in vivo and in vitro based on the transfer of energy from adonor fluorophore to an acceptor fluorophore. Selvin, Nat. Struct.Biol., 7:730-34 (2000). Energy transfer takes place only if the donorfluorophore is in sufficient proximity to the acceptor fluorophore. In atypical FRET experiment, two proteins or two sites on a single proteinare labeled with different fluorescent probes. One of the probes, thedonor probe, is excited to a higher energy state by incident light of aspecified wavelength. The donor probe then transmits its energy to thesecond probe, the acceptor probe, resulting in a reduction in thedonor's fluorescence intensity and an increase in the acceptor'sfluorescence emission. To measure the extent of energy transfer, thedonor's intensity in a sample labeled with donor and acceptor probes iscompared with its intensity in a sample labeled with donor probe only.Optionally, acceptor intensity is compared in donor/acceptor andacceptor only samples. Suitable probes are known in the art and include,for example, membrane permeant dyes, such as fluorescein and rhodamine,organic dyes, such as the cyanine dyes, and lanthanide atoms. Selvin,supra. Methods and instrumentation for detecting and measuring energytransfer are also known in the art. Selvin, supra.

FRET-based techniques suitable for detecting and measuringprotein-protein interactions in individual cells are also known in theart. For example, donor photobleaching fluorescence resonance energytransfer (pbFRET) microscopy and fluorescence lifetime imagingmicroscopy (FLIM) may be used to detect the dimerization of cell surfacereceptors. Selvin, supra; Gadella & Jovin, J. Cell Biol., 129:1543-58(1995).

For example, antibodies against VEGFR2 and VE-cadherin are directlylabeled with two different fluorophores. Endothelial cell lysates arecontacted with the differentially labeled antibodies, which act asdonors and acceptors for FRET in the presence of VEGFR2 and VE-cadherinheterodimer. Energy transfer is detected and the presence ofheterodimers is determined if the labels are found to be in closeproximity.

4) Assays Using Releasable Molecular Tags

In some preferred embodiments, assays using releasable molecular tagsare used to detect protein-protein interactions, such as protein dimerformation, and measure multiple proteins simultaneously.

Many advantages are provided by measuring dimer populations usingreleasable molecular tags (such as with eTag™ assays, as describedbelow), including (1) separation of released molecular tags from anassay mixture provides greatly reduced background and a significant gainin sensitivity; and (2) the use of molecular tags that are speciallydesigned for ease of separation and detection provides a convenientmultiplexing capability so that multiple receptor complex components maybe readily measured simultaneously in the same assay. Assays employingsuch tags can have a variety of forms and are disclosed in the followingreferences: Singh et U.S. Pat. No. 6,627,400; U.S. patent publicationsSingh et al., 2002/0013126; and 2003/0170915, and Williams et al.,2002/0146726; and Chan-Hui et al., International patent publication WO2004/011900, all of which are incorporated herein by reference. Forexample, a wide variety of separation techniques may be employed thatcan distinguish molecules based on one or more physical, chemical, oroptical differences among molecules being separated including but notlimited to electrophoretic mobility, molecular weight, shape,solubility, pKa, hydrophobicity, charge, charge/mass ratio, polarity, orthe like. In one aspect, molecular tags in a plurality or set differ inelectrophoretic mobility and optical detection characteristics and areseparated by electrophoresis. In another aspect, molecular tags in aplurality or set may differ in molecular weight, shape, solubility, pKa,hydrophobicity, charge, polarity, and are separated by normal phase orreverse phase HPLC, ion exchange HPLC, capillary electrochromatography,mass spectroscopy, gas phase chromatography, or like technique.

Sets of molecular tags are provided that are separated into distinctbands or peaks by a separation technique after they are released frombinding compounds. Identification and quantification of such peaksprovides a measure or profile of the kinds and amounts of receptordimers. Molecular tags within a set may be chemically diverse; however,for convenience, sets of molecular tags are usually chemically related.For example, they may all be peptides, or they may consist of differentcombinations of the same basic building blocks or monomers, or they maybe synthesized using the same basic scaffold with different substituentgroups for imparting different separation characteristics, as describedmore fully below. The number of molecular tags in a plurality may varydepending on several factors including the mode of separation employed,the labels used on the molecular tags for detection, the sensitivity ofthe binding moieties, the efficiency with which the cleavable linkagesare cleaved, and the like. In one aspect, the number of molecular tagsin a plurality for measuring populations of receptor dimers is in therange of from 2 to 10. In other aspects, the size of the plurality maybe in the range of from 2 to 8, 2 to 6, 2 to 4, or 2 to 3.

Protein dimers (e.g., VEGFR, Tie dimers) may be detected in assayshaving homogeneous formats or a non-homogeneous, i.e. heterogeneous,format. In a homogeneous format, no step is required to separate bindingcompounds specifically bound to target complexes from unbound bindingcompounds. In a preferred embodiment, homogeneous formats employ reagentpairs comprising (i) one or more binding compounds with releasablemolecular tags and (ii) at least one cleaving probe that is capable ofgenerating an active species that reacts with and releases moleculartags within an effective proximity of the cleaving probe.

Protein dimers may also be detected by assays employing a heterogeneousformat. Heterogeneous techniques normally involve a separation step,where intracellular complexes having binding compounds specificallybound are separated from unbound binding compounds, and optionally,other sample components, such as proteins, membrane fragments, and thelike. Separation can be achieved in a variety of ways, such as employinga reagent bound to a solid support that distinguishes betweencomplex-bound and unbound binding compounds. The solid support may be avessel wall, e.g., microtiter well plate well, capillary, plate, slide,beads, including magnetic beads, liposomes, or the like. The primarycharacteristics of the solid support are that it (1) permits segregationof the bound and unbound binding compounds and (2) does not interferewith the formation of the binding complex, or the other operations inthe determination of receptor dimers. Usually, in fixed samples, unboundbinding compounds are removed simply by washing.

With detection using molecular tags in a heterogeneous format, afterwashing, a sample may be combined with a solvent into which themolecular tags are to be released. Depending on the nature of thecleavable bond and the method of cleavage, the solvent may include anyadditional reagents for the cleavage. Where reagents for cleavage arenot required, the solvent conveniently may be a separation buffer, e.g.an electrophoretic separation medium. For example, where the cleavablelinkage is photolabile or cleavable via an active species generated by aphotosensitizer, the medium may be irradiated with light of appropriatewavelength to release the molecular tags into the buffer.

In either format, if the assay reaction conditions interfere with theseparation technique employed, it may be necessary to remove, orexchange, the assay reaction buffer prior to cleavage and separation ofthe molecular tags. For example, in some embodiments, assay conditionsinclude salt concentrations (e.g. required for specific binding) thatdegrade separation performance when molecular tags are separated on thebasis of electrophoretic mobility. In such embodiments, an assay bufferis replaced by a separation buffer, or medium, prior to release andseparation of the molecular tags.

Assays employing releasable molecular tags and cleaving probes can bemade in many different formats and configurations depending on thecomplexes that are detected or measured. Based on the presentdisclosure, it is a design choice for one of ordinary skill in the artto select the numbers and specificities of particular binding compoundsand cleaving probes.

In one aspect of the invention, the use of releasable molecular tags tomeasure components of signaling pathways is shown diagrammatically inFIGS. 4A-H. The operation of such assays to provide ratiometricmeasurements is illustrated in FIG. 4A. Effector protein (10) exists intwo states in a cell, one having a post-translational modification, e.g.such as a phosphate group (12), and the other not having such apost-translational modification. Reagents (14) of the invention,comprising cleaving probes (18) (in this illustration havingphotosensitizer “PS” attached) and binding compounds (16 and 17), aremixed (19) with a sample containing both the activated and inactivatedforms of effector protein (10) under conditions that permit the specificbinding of cleaving probes (18) and binding compounds (16 and 17) totheir respective antigenic determinants on the activated and inactivatedforms of effector protein (10) resulting in the formation of eithercomplex (21) or complex (23). After binding, and optionally washing orbuffer exchange, cleaving probes (18) are activated to generate anactive species that, e.g. in the case of singlet oxygen, diffuses outfrom a photosensitizers to an effective proximity (20). Cleavablelinkages within this proximity are cleaved and molecular tags arereleased (22). Released molecular tags (22) are then separated (25) anda separation profile (28), such as an electropherogram, is produced, inwhich peak (24) is identified and correlated to molecular tag, “mT₁” andpeak (26) is identified and correlated to molecular tag, “mT₂.” In oneaspect, a ratiometric measure of activated effector protein (10) isprovided as the ratio of areas of peaks (24) and (26).

A method of measuring signaling complexes comprising heterodimers isillustrated in FIG. 4B. Signaling complex (100) forms by the binding ofproteins (104) and (102), e.g. Akt and PDK 1. Reagents (122) of theinvention, comprising cleaving probes (108) (in this illustration havingphotosensitizer “PS” attached) and binding compounds (106 and 107), aremixed (109) with a sample containing complex (100) under conditions thatpermit the specific binding (112) of cleaving probes (108) and bindingcompounds (106 and 107) to their respective antigenic determinants oncomplex (100) that are on different proteins of the complex. Afterbinding, and optionally washing or buffer exchange, cleaving probes(108) are activated to generate an active species that, e.g. in the caseof singlet oxygen, diffuses out from a photosensitizers to an effectiveproximity (110). Cleavable linkages within this proximity are cleavedand molecular tags are released (114). Released molecular tags (116) arethen separated (117) and a separation profile (120), such as anelectropherogram, is produced, in which peak (118) is identified andcorrelated to molecular tag, “mT₁” and peak (124) is identified andcorrelated with molecular tag, “mT₂.” By employing additional bindingcompounds and molecular tags, additional complexes may be measured. Aswith the ratiometric measure of an activated effector protein, theamount of heterodimeric complexes may be provided as a ratio of peakareas. FIG. 4D illustrates the analogous measurements for cell surfacereceptors that form heterodimers in cell surface membrane (161).

Homodimeric as well as heterodimeric complexes may be measured asillustrated in FIG. 4C. As above, an assay may comprise three reagents(128): cleaving probes (134), first binding compound (130), and secondbinding compound (132). First binding compound (130) and cleaving probe(134) are constructed to be specific for the same antigenic determinant(135) on protein (138) that exists (140) in a sample as either ahomodimer (136) or a monomer (138). After reagents (128) are combinedwith a sample under conditions that promote the formation of stablecomplexes between the reagents and their respective targets, multiplecomplexes (142 through 150) form in the assay mixture. Because cleavingprobe (134) and binding compound (130) are specific for the sameantigenic determinant (135), four different combinations (144 through150) of reagents may form complexes with homodimers. Of the complexes inthe assay mixture, only those (143) with both a cleaving probe (134) andat least one binding compound will contribute released molecular tags(151) for separation and detection (154). In this embodiment, the sizeof peak (153) is proportional to the amount of homodimer in the assaymixture, while the size of peak (152) is proportional to the totalamount of protein (138) in the assay mixture, both in monomeric form(142) or in homodimeric form (146 and 148).

Another aspect of the invention is illustrated in FIGS. 4E and 4F, whichprovides for the simultaneous detection or measurement of multiplecomplexes, dimers, and activated effector proteins in a cellular sample.Cells (160), which may be from a sample from in vitro cultures or from aspecimen of patient tissue, are lysed (172) to form lysate (174) inwhich cellular components are rendered accessible, such componentsincluding molecular complexes associated with the cell membrane (173),and/or within the cytosol (179), and/or within the cell nucleus.Complexes associated with signaling pathways include, but are notlimited to, surface receptor complexes, such as receptor dimers (162 or170), receptor complexes including adaptor or scaffold molecules ofvarious types (162, 168, or 170), dimers and higher order complexes ofintracellular proteins (164), phosphorylation sites of proteins in suchcomplexes (166), phosphorylated effector proteins (163), and the like.After lysing, the resulting lysate (174) is combined with assay reagents(176) that include multiple cleaving probes (175) and multiple bindingcompounds (177). Assay conditions are selected (178) that allow reagents(176) to specifically bind to their respective targets, so that uponactivation cleavable linkages within the effective proximity (180) ofthe cleavage-inducing moieties are cleaved and molecular tags arereleased (182). Also illustrated are intracellular complexes, e.g.signaling complexes (181), receptor dimers (183), and effector proteins(185). As above, after cleavage, the released molecular tags areseparated (184) and identified in a separation profile (186), such as anelectropherogram, and based on the number and quantities of moleculartags measured, a profile is obtained of the selected molecularcomplexes, protein dimers, and/or effector proteins in the cells of thesample.

One skilled in the art would understand that dimers may be measured ineither lysates of cells or tissues, or in fixed samples whose membraneshave been permeabilized or removed by the fixing process. In such cases,binding compounds may be specific for either extracellular orintracellular domains of cell surface membrane receptors.

FIGS. 4G and 4H illustrate an embodiment of the invention for measuringreceptor complexes in fixed or frozen tissue samples. Fixed tissuesample (1000), e.g. a formalin-fixed paraffin-embedded sample, is slicedto provide a section (1004) using a microtome, or like instrument, whichafter placing on surface (1006), which may be a microscope slide, it isde-waxed and re-hydrated for application of assay reagents. Enlargement(1007) shows portion (1008) of section (1004) on portion (1014) ofmicroscope slide (1006). Receptor dimer molecules (1018) are illustratedas embedded in the remnants of membrane structure (1016) of the fixedsample. In accordance with this aspect of the invention, cleaving probeand binding compounds are incubated with the fixed sample so that theybind to their target molecules. For example, cleaving probes (1012)(illustrated in the figure as an antibody having a photosensitizer(“PS”) attached) and first binding compound (1010) (illustrated as anantibody having molecular tag “mT₁” attached) specifically bind toreceptor (1011) common to all of the dimers shown, second bindingcompound (1017) (with “mT₂”) specifically binds to receptor (1015), andthird binding compound (1019) (with “mT₃”) specifically binds toreceptor (1013). After washing to remove binding compounds and cleavingprobe that are not specifically bound to their respective targetmolecules, buffer (1024) (referred to as “illumination buffer” in thefigure) is added. For convenience, buffer (1024) may be contained onsection (1004), or a portion thereof, by creating a hydrophobic barrieron slide (1006), e.g. with a wax pen. After illumination ofphotosensitizers and release of molecular tags (1026), buffer (1024) nowcontaining release molecular tags (1025) is transferred to a separationdevice, such as a capillary electrophoresis instrument, for separation(1028) and identification of the released molecular tags in, forexample, electropherogram (1030).

Measurements made directly on tissue samples, particularly asillustrated in FIGS. 4G and 4H, may be normalized by includingmeasurements on cellular or tissue targets that are representative ofthe total cell number in the sample and/or the numbers of particularsubtypes of cells in the sample. The additional measurement may bepreferred, or even necessary, because of the cellular and tissueheterogeneity in patient samples, particularly tumor samples, which maycomprise substantial fractions of normal cells. For example, in FIG. 4H,values for the total amount of receptor (1011) may be given as a ratioof the following two measurements: area of peak (1032) of molecular tag(“mT₁”) and the area of a peak corresponding to a molecular tagcorrelated with a cellular or tissue component common to all the cellsin the sample, e.g. tubulin, or the like. Accordingly, detection methodsbased on releasable molecular tags may include an additional step ofproviding a binding compound (with a distinct molecular tag) specificfor a normalization protein, such as tubulin.

A preferred embodiment for measuring relative amounts of receptor dimerscontaining a common component receptor is illustrated in FIG. 6. In thisassay design, two different receptor dimers (“1-2” (240) and “2-3”(250)) each having a common component, “2,” may be measuredratiometrically with respect to the common component. An assay design isshown for measuring receptor heterodimer (240) comprising receptor “1”(222) and receptor “2” (220) and receptor heterodimer (250) comprisingreceptor “2” (220) and receptor “3” (224). A key feature of thisembodiment is that cleaving probe (227) is made specific for the commonreceptor of the pair of heterodimers. Binding compound (228) specificfor receptor “2” provides a signal (234) related to the total amount ofreceptor “2” in the assay, whereas binding compound (226) specific forreceptor “1” and binding compound (230) specific for receptor “3”provide signals (232 and 236, respectively) related only to the amountof receptor “1” and receptor “3” present as heterodimers with receptor“2,” respectively. The design of FIG. 6 may be generalized to more thantwo receptor complexes that contain a common component by simply addingbinding compounds specific for the components of the additionalcomplexes.

A. Binding Compounds and Molecular Tags

As mentioned above, mixtures containing pluralities of different bindingcompounds may be provided, wherein each different binding compound hasone or more molecular tags attached through cleavable linkages. Thenature of the binding compound, cleavable linkage and molecular tag mayvary widely. A binding compound may comprise an antibody bindingcomposition, an antibody, a peptide, a peptide or non-peptide ligand fora cell surface receptor, a protein, an oligonucleotide, anoligonucleotide analog, such as a peptide nucleic acid, a lectin, or anyother molecular entity that is capable of specific binding or stablecomplex formation with an analyte of interest, such as a complex ofproteins. In one aspect, a binding compound, which can be represented bythe formula below, comprises one or more molecular tags attached to abinding moiety.

B-(L-E)_(k)

wherein B is binding moiety; L is a cleavable linkage; and E is amolecular tag. In homogeneous assays, cleavable linkage, L, may be anoxidation-labile linkage, and more preferably, it is a linkage that maybe cleaved by singlet oxygen. The moiety “-(L-E)_(k)” indicates that asingle binding compound may have multiple molecular tags attached viacleavable linkages. In one aspect, k is an integer greater than or equalto one, but in other embodiments, k may be greater than several hundred,e.g. 100 to 500, or k is greater than several hundred to as many asseveral thousand, e.g. 500 to 5000. Usually each of the plurality ofdifferent types of binding compound has a different molecular tag, E.Cleavable linkages, e.g. oxidation-labile linkages, and molecular tags,E, are attached to B by way of conventional chemistries.

Preferably, B is an antibody binding composition that specifically bindsto a target, such as a predetermined antigenic determinant of a targetprotein, such as a cell surface receptor. Such compositions are readilyformed from a wide variety of commercially available antibodies, bothmonoclonal and polyclonal, specific for proteins of interest. Inparticular, antibodies specific for epidermal growth factor receptorsare disclosed in the following patents, all of which are incorporated byreferences: U.S. Pat. Nos. 5,677,171; 5,772,997; 5,968,511; 5,480,968;5,811,098. U.S. Pat. No. 6,488,390, incorporated herein by reference,discloses antibodies specific for a G-protein coupled receptor, CCR4.U.S. Pat. No. 5,599,681, incorporated herein by reference, disclosesantibodies specific for phosphorylation sites of proteins. Commercialvendors, such as Cell Signaling Technology (Beverly, Mass.), BiosourceInternational (Camarillo, Calif.), and Upstate (Charlottesville, Va.),also provide monoclonal and polyclonal antibodies specific for manyreceptors.

Cleavable linkage, L, can be virtually any chemical linking group thatmay be cleaved under conditions that do not degrade the structure oraffect detection characteristics of the released molecular tag, E.Whenever a cleaving probe is used in a homogeneous assay format,cleavable linkage, L, is cleaved by a cleavage agent generated by thecleaving probe that acts over a short distance so that only cleavablelinkages in the immediate proximity of the cleaving probe are cleaved.Typically, such an agent must be activated by making a physical orchemical change to the reaction mixture so that the agent produces ashort lived active species that diffuses to a cleavable linkage toeffect cleavage. In a homogeneous format, the cleavage agent ispreferably attached to a binding moiety, such as an antibody, thattargets prior to activation the cleavage agent to a particular site inthe proximity of a binding compound with releasable molecular tags. Insuch embodiments, a cleavage agent is referred to herein as acleavage-inducing moiety, which is discussed more fully below.

In a non-homogeneous format, because specifically bound bindingcompounds are separated from unbound binding compounds, a widerselection of cleavable linkages and cleavage agents are available foruse. Cleavable linkages may not only include linkages that are labile toreaction with a locally acting reactive species, such as hydrogenperoxide, singlet oxygen, or the like, but also linkages that are labileto agents that operate throughout a reaction mixture, such asbase-labile linkages, photocleavable linkages, linkages cleavable byreduction, linkages cleaved by oxidation, acid-labile linkages, peptidelinkages cleavable by specific proteases, and the like. Referencesdescribing many such linkages include Greene and Wuts, Protective Groupsin Organic Synthesis, Second Edition (John Wiley & Sons, New York,1991); Hermanson, Bioconjugate Techniques (Academic Press, New York,1996); and Still et al., U.S. Pat. No. 5,565,324.

In one aspect, commercially available cleavable reagent systems may beemployed with the invention. For example, a disulfide linkage may beintroduced between an antibody binding composition and a molecular tagusing a heterofunctional agent such as N-succinimidyl3-(2-pyridyldithio)propionate (SPDP),succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT), orthe like, available from vendors such as Pierce Chemical Company(Rockford, Ill.). Disulfide bonds introduced by such linkages can bebroken by treatment with a reducing agent, such as dithiothreitol (DTT),dithioerythritol (DTE), 2-mercaptoethanol, sodium borohydride, or thelike. Typical concentrations of reducing agents to effect cleavage ofdisulfide bonds are in the range of from 10 to 100 mM. An oxidativelylabile linkage may be introduced between an antibody binding compositionand a molecular tag using the homobifunctional NHS ester cross-linkingreagent, disuccinimidyl tartarate (DST) (available from Pierce) thatcontains central cis-diols that are susceptible to cleavage with sodiumperiodate (e.g., 15 mM periodate at physiological pH for 4 hours).Linkages that contain esterified spacer components may be cleaved withstrong nucleophilic agents, such as hydroxylamine, e.g., 0.1 Nhydroxylamine, pH 8.5, for 3-6 hours at 37° C. Such spacers can beintroduced by a homobifunctional cross-linking agent such as ethyleneglycol bis(succinimidylsuccinate)(EGS) available from Pierce (Rockford,Ill.). A base labile linkage can be introduced with a sulfone group.Homobifunctional cross-linking agents that can be used to introducesulfone groups in a cleavable linkage includebis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES), and4,4-difluoro-3,3-dinitrophenylsulfone (DFDNPS). Exemplary basicconditions for cleavage include 0.1 M sodium phosphate, adjusted to pH11.6 by addition of Tris base, containing 6 M urea, 0.1% SDS, and 2 mMDTT, with incubation at 37° C. for 2 hours. Photocleavable linkagesinclude those disclosed in Rothschild et al., U.S. Pat. No. 5,986,076.

When L is oxidation labile, L may be a thioether or its selenium analog;or an olefin, which contains carbon-carbon double bonds, whereincleavage of a double bond to an oxo group, releases the molecular tag,E. Illustrative oxidation labile linkages are disclosed in Singh et al.,U.S. Pat. No. 6,627,400; and U.S. patent publications Singh et al.,2002/0013126; and 2003/0170915, and in Willner et al., U.S. Pat. No.5,622,929, all of which are incorporated herein by reference.

Molecular tag, E, in the present invention may comprise an electrophorictag as described in the following references when separation ofpluralities of molecular tags are carried out by gas chromatography ormass spectrometry: Zhang et al., Bioconjugate Chem., 13: 1002-1012(2002); Giese, Anal. Chem., 2: 165-168 (1983); and U.S. Pat. Nos.4,650,750; 5,360,819; 5,516,931; 5,602,273; and the like.

Molecular tag, E, is preferably a water-soluble organic compound that isstable with respect to the active species, especially singlet oxygen,and that includes a detection or reporter group. Otherwise, E may varywidely in size and structure. In one aspect, E has a molecular weight inthe range of from about 50 to about 2500 daltons, more preferably, fromabout 50 to about 1500 daltons. Preferred structures of E are describedmore fully below. E may comprise a detection group for generating anelectrochemical, fluorescent, or chromogenic signal. In embodimentsemploying detection by mass, E may not have a separate moiety fordetection purposes. Preferably, the detection group generates afluorescent signal.

B. Attaching Molecular Tags to Binding Moieties

Extensive guidance can be found in the literature for covalently linkingmolecular tags to binding compounds, such as antibodies, e.g. Hermanson,Bioconjugate Techniques, (Academic Press, New York, 1996), and the like.In one aspect of the invention, one or more molecular tags are attacheddirectly or indirectly to common reactive groups on a binding compound.Common reactive groups include amine, thiol, carboxylate, hydroxyl,aldehyde, ketone, and the like, and may be coupled to molecular tags bycommercially available cross-linking agents, e.g. Hermanson (citedabove); Haugland, Handbook of Fluorescent Probes and Research Products,Ninth Edition (Molecular Probes, Eugene, Oreg., 2002). In oneembodiment, an NHS-ester of a molecular tag is reacted with a free amineon the binding compound.

In another embodiment illustrated in FIG. 5A, binding compounds comprisea biotinylated antibody (200) as a binding moiety. Molecular tags areattached to binding moiety (200) by way of avidin or streptavidin bridge(206). Preferably, in operation, binding moiety (200) is first reactedwith a target complex, after which avidin or streptavidin is added (204)to form antibody-biotin-avidin complex (205). To such complexes (205)are added (208) biotinylated molecular tags (210) to form bindingcompound (212).

In still another embodiment illustrated in FIG. 5B, binding compoundscomprise an antibody (214) derivatized with a multi-functional moiety(216) that contains multiple functional groups (218) that are reacted(220) molecular tag precursors to give a final binding compound havingmultiple molecular tags (222) attached. Exemplary multi-functionalmoieties include aminodextran, and like materials.

Once each of the binding compounds is separately derivatized by adifferent molecular tag, it is pooled with other binding compounds toform a plurality of binding compounds. Usually, each different kind ofbinding compound is present in a composition in the same proportion;however, proportions may be varied as a design choice so that one or asubset of particular binding compounds are present in greater or lowerproportion depending on the desirability or requirements for aparticular embodiment or assay. Factors that may affect such designchoices include, but are not limited to, antibody affinity and avidityfor a particular target, relative prevalence of a target, fluorescentcharacteristics of a detection moiety of a molecular tag, and the like.

C. Separation of Released Molecular Tags

Molecular tags within a plurality are selected so that each has a uniqueseparation characteristic and/or a unique optical property with respectto the other members of the same plurality. In one aspect, thechromatographic or electrophoretic separation characteristic isretention time under set of standard separation conditions conventionalin the art, e.g. voltage, column pressure, column type, mobile phase,electrophoretic separation medium, or the like. In another aspect, theoptical property is a fluorescence property, such as emission spectrum,fluorescence lifetime, fluorescence intensity at a given wavelength orband of wavelengths, or the like. Preferably, the fluorescence propertyis fluorescence intensity. For example, each molecular tag of aplurality may have the same fluorescent emission properties, but eachwill differ from one another by virtue of a unique retention time. Onthe other hand, or two or more of the molecular tags of a plurality mayhave identical migration, or retention, times, but they will have uniquefluorescent properties, e.g. spectrally resolvable emission spectra, sothat all the members of the plurality are distinguishable by thecombination of molecular separation and fluorescence measurement.

Preferably, released molecular tags are detected by electrophoreticseparation and the fluorescence of a detection group. In suchembodiments, molecular tags having substantially identical fluorescenceproperties have different electrophoretic mobilities so that distinctpeaks in an electropherogram are formed under separation conditions.Preferably, pluralities of molecular tags of the invention are separatedby conventional capillary electrophoresis apparatus, either in thepresence or absence of a conventional sieving matrix. Exemplarycapillary electrophoresis apparatus include Applied Biosystems (FosterCity, Calif.) models 310, 3100 and 3700; Beckman (Fullerton, Calif.)model P/ACE MDQ; Amersham Biosciences (Sunnyvale, Calif.) MegaBACE 1000or 4000; SpectruMedix genetic analysis system; and the like.Electrophoretic mobility is proportional to q/M^(2/3), where q is thecharge on the molecule and M is the mass of the molecule. Desirably, thedifference in mobility under the conditions of the determination betweenthe closest electrophoretic labels will be at least about 0.001, usually0.002, more usually at least about 0.01, and may be 0.02 or more.Preferably, in such conventional apparatus, the electrophoreticmobilities of molecular tags of a plurality differ by at least onepercent, and more preferably, by at least a percentage in the range offrom 1 to 10 percent. Molecular tags are identified and quantified byanalysis of a separation profile, or more specifically, anelectropherogram, and such values are correlated with the amounts andkinds of receptor dimers present in a sample. For example, during orafter electrophoretic separation, the molecular tags are detected oridentified by recording fluorescence signals and migration times (ormigration distances) of the separated compounds, or by constructing achart of relative fluorescent and order of migration of the moleculartags (e.g., as an electropherogram). Preferably, the presence, absence,and/or amounts of molecular tags are measured by using one or morestandards as disclosed by Williams et al., U.S. patent publication2003/0170734A1, which is incorporated herein by reference.

Pluralities of molecular tags may also be designed for separation bychromatography based on one or more physical characteristics thatinclude but are not limited to molecular weight, shape, solubility, pKa,hydrophobicity, charge, polarity, or the like, e.g. as disclosed in U.S.patent publication 2003/0235832, which is incorporated by reference. Achromatographic separation technique is selected based on parameterssuch as column type, solid phase, mobile phase, and the like, followedby selection of a plurality of molecular tags that may be separated toform distinct peaks or bands in a single operation. Several factorsdetermine which HPLC technique is selected for use in the invention,including the number of molecular tags to be detected (i.e. the size ofthe plurality), the estimated quantities of each molecular tag that willbe generated in the assays, the availability and ease of synthesizingmolecular tags that are candidates for a set to be used in multiplexedassays, the detection modality employed, and the availability,robustness, cost, and ease of operation of HPLC instrumentation,columns, and solvents. Generally, columns and techniques are favoredthat are suitable for analyzing limited amounts of sample and thatprovide the highest resolution separations. Guidance for making suchselections can be found in the literature, e.g. Snyder et al., PracticalHPLC Method Development, (John Wiley & Sons, New York, 1988); Millner,“High Resolution Chromatography: A Practical Approach”, OxfordUniversity Press, New York (1999), Chi-San Wu, “Column Handbook for SizeExclusion Chromatography”, Academic Press, San Diego (1999), and Oliver,“HPLC of Macromolecules: A Practical Approach, Oxford University Press”,Oxford, England (1989). In particular, procedures are available forsystematic development and optimization of chromatographic separationsgiven conditions, such as column type, solid phase, and the like, e.g.Haber et Chromatogr. Sci., 38: 386-392 (2000); Outinen et al., Eur. J.Pharm. Sci., 6: 197-205 (1998); Lewis et al., J. Chromatogr., 592:183-195 and 197-208 (1992); and the like. An exemplary HPLCinstrumentation system suitable for use with the present invention isthe Agilent 1100 Series HPLC system (Agilent Technologies, Palo Alto,Calif.).

In one aspect, molecular tag, E, is (M, D), where M is amobility-modifying moiety and D is a detection moiety. The notation “(M,D)” is used to indicate that the ordering of the M and D moieties may besuch that either moiety can be adjacent to the cleavable linkage, L.That is, “B-L-(M, D)” designates binding compound of either of twoforms: “B-L-M-D” or “B-L-D-M.”

Detection moiety, D, may be a fluorescent label or dye, a chromogeniclabel or dye, an electrochemical label, or the like. Preferably, D is afluorescent dye. Exemplary fluorescent dyes for use with the inventioninclude water-soluble rhodamine dyes, fluoresceins,4,7-dichlorofluoresceins, benzoxanthene dyes, and energy transfer dyes,disclosed in the following references: Handbook of Molecular Probes andResearch Reagents, 8^(th) ed., (Molecular Probes, Eugene, 2002); Lee etal., U.S. Pat. No. 6,191,278; Lee et al., U.S. Pat. No. 6,372,907;Menchen et al., U.S. Pat. No. 6,096,723; Lee et al., U.S. Pat. No.5,945,526; Lee et al., Nucleic Acids Research, 25: 2816-2822 (1997);Hobb, Jr., U.S. Pat. No. 4,997,928; Khanna et al., U.S. Pat. No.4,318,846; and the like. Preferably, D is a fluorescein or a fluoresceinderivative.

D. Cleavage-Inducing Moiety Producing Active Species

A cleavage-inducing moiety, or cleaving agent, is a group that producesan active species that is capable of cleaving a cleavable linkage,preferably by oxidation. Preferably, the active species is a chemicalspecies that exhibits short-lived activity so that its cleavage-inducingeffects are only in the proximity of the site of its generation. Eitherthe active species is inherently short lived, so that it will not createsignificant background because beyond the proximity of its creation, ora scavenger is employed that efficiently scavenges the active species,so that it is not available to react with cleavable linkages beyond ashort distance from the site of its generation. Illustrative activespecies include singlet oxygen, hydrogen peroxide, NADH, and hydroxylradicals, phenoxy radical, superoxide, and the like. Illustrativequenchers for active species that cause oxidation include polyenes,carotenoids, vitamin E, vitamin C, amino acid-pyrrole N-conjugates oftyrosine, histidine, and glutathione, and the like, e.g. Beutner et al.,Meth. Enzymol., 319: 226-241 (2000).

An important consideration in designing assays employing acleavage-inducing moiety and a cleavable linkage is that they not be sofar removed from one another when bound to a receptor complex that theactive species generated by the cleavage-inducing moiety cannotefficiently cleave the cleavable linkage. In one aspect, cleavablelinkages preferably are within 1000 nm, and preferably within 20-200 nm,of a bound cleavage-inducing moiety. More preferably, forphotosensitizer cleavage-inducing moieties generating singlet oxygen,cleavable linkages are within about 20-100 nm of a photosensitizer in areceptor complex. The range within which a cleavage-inducing moiety caneffectively cleave a cleavable linkage (that is, cleave enough moleculartag to generate a detectable signal) is referred to herein as its“effective proximity.” One of ordinary skill in the art recognizes thatthe effective proximity of a particular sensitizer may depend on thedetails of a particular assay design and may be determined or modifiedby routine experimentation.

A sensitizer is a compound that can be induced to generate a reactiveintermediate, or species, usually singlet oxygen. Preferably, asensitizer used in accordance with the invention is a photosensitizer.Other sensitizers included within the scope of the invention arecompounds that on excitation by heat, light, ionizing radiation, orchemical activation will release a molecule of singlet oxygen. The bestknown members of this class of compounds include the endoperoxides suchas 1,4-biscarboxyethyl-1,4-naphthalene endoperoxide,9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating or direct absorption of light bythese compounds releases singlet oxygen. Further sensitizers aredisclosed in the following references: Di Mascio et al., FEBS Lett.,355: 287 (1994)(peroxidases and oxygenases); Kanofsky, J.Biol. Chem.258: 5991-5993 (1983)(lactoperoxidase); Pierlot et al., Meth. Enzymol.,319: 3-20 (2000)(thermal lysis of endoperoxides); and the like.Attachment of a binding agent to the cleavage-inducing moiety may bedirect or indirect, covalent or non-covalent and can be accomplished bywell-known techniques, commonly available in the literature. See, forexample, “Immobilized Enzymes,” Ichiro Chibata, Halsted Press, New York(1978); Cuatrecasas, J. Biol. Chem., 245:3059 (1970).

As mentioned above, the preferred cleavage-inducing moiety in accordancewith the present invention is a photosensitizer that produces singletoxygen. As used herein, “photosensitizer” refers to a light-adsorbingmolecule that when activated by light converts molecular oxygen intosinglet oxygen. Photosensitizers may be attached directly or indirectly,via covalent or non-covalent linkages, to the binding agent of aclass-specific reagent. Guidance for constructing of such compositions,particularly for antibodies as binding agents, is available in theliterature, e.g. in the fields of photodynamic therapy,immunodiagnostics, and the like. The following are exemplary references:Ullman, et al., Proc. Natl. Acad. Sci. USA 91, 5426-5430 (1994); Stronget al., Ann. New York Acad. Sci., 745: 297-320 (1994); Yarmush et al.,Crit. Rev. Therapeutic Drug Carrier Syst., 10: 197-252 (1993); Pease etal., U.S. Pat. No. 5,709,994; Ullman et al., U.S. Pat. No. 5,340,716;Ullman et al., U.S. Pat. No. 6,251,581; McCapra, U.S. Pat. No.5,516,636; and the like.

A large variety of light sources are available to photo-activatephotosensitizers to generate singlet oxygen. Both polychromatic andmonochromatic sources may be used as long as the source is sufficientlyintense to produce enough singlet oxygen in a practical time duration.The length of the irradiation is dependent on the nature of thephotosensitizer, the nature of the cleavable linkage, the power of thesource of irradiation, and its distance from the sample, and so forth.In general, the period for irradiation may be less than about amicrosecond to as long as about 10 minutes, usually in the range ofabout one millisecond to about 60 seconds. The intensity and length ofirradiation should be sufficient to excite at least about 0.1% of thephotosensitizer molecules, usually at least about 30% of thephotosensitizer molecules and preferably, substantially all of thephotosensitizer molecules. Exemplary light sources include, by way ofillustration and not limitation, lasers such as, e.g., helium-neonlasers, argon lasers, YAG lasers, He/Cd lasers, and ruby lasers;photodiodes; mercury, sodium and xenon vapor lamps; incandescent lampssuch as, e.g., tungsten and tungsten/halogen; flashlamps; and the like.By way of example, a photoactivation device disclosed in Bjornson etal., International patent publication WO 03/051669 is employed. Briefly,the photoactivation device is an array of light emitting diodes (LEDs)mounted in housing that permits the simultaneous illumination of all thewells in a 96-well plate. A suitable LED for use in the presentinvention is a high power GaAIAs IR emitter, such as model OD-880Wmanufactured by OPTO DIODE CORP. (Newbury Park, Calif.).

Examples of photosensitizers that may be utilized in the presentinvention are those that have the above properties and are enumerated inthe following references: Singh and Ullman, U.S. Pat. No. 5,536,834; Liet al., U.S. Pat. No. 5,763,602; Martin et al., Methods Enzymol., 186:635-645 (1990); Yarmush et al., Crit. Rev. Therapeutic Drug CarrierSyst., 10: 197-252 (1993); Pease et al., U.S. Pat. No. 5,709,994; Ullmanet al., U.S. Pat. No. 5,340,716; Ullman et al., U.S. Pat. No. 6,251,581;McCapra, U.S. Pat. No. 5,516,636; Thetford, European patent publ.0484027; Sessler et al., SPIE, 1426: 318-329 (1991); Magda et al., U.S.Pat. No. 5,565,552; Roelant, U.S. Pat. No. 6,001,673; and the like.

As with sensitizers, in certain embodiments, a photosensitizer may beassociated with a solid phase support by being covalently ornon-covalently attached to the surface of the support or incorporatedinto the body of the support. In general, the photosensitizer isassociated with the support in an amount necessary to achieve thenecessary amount of singlet oxygen. Generally, the amount ofphotosensitizer is determined empirically.

In one embodiment, a photosensitizer is incorporated into a latexparticle to form photosensitizer beads, e.g. as disclosed by Pease etal., U.S. Pat. No. 5,709,994; Pollner, U.S. Pat. No. 6,346,384; andPease et al., PCT publication WO 01/84157. Alternatively,photosensitizer beads may be prepared by covalently attaching aphotosensitizer, such as rose bengal, to 0.5 micron latex beads by meansof chloromethyl groups on the latex to provide an ester linking group,as described in J. Amer. Chem. Soc., 97: 3741 (1975). Use of suchphotosensitizer beads is illustrated in FIGS. 5C and 5D. As described inFIG. 4B for heteroduplex detection, complexes (230) are formed aftercombining reagents (1122) with a sample (1100). This reaction may becarried out, for example, in a conventional 96-well or 384-wellmicrotiter plate, or the like, having a filter membrane that forms onewall, e.g. the bottom, of the wells that allows reagents to be removedby the application of a vacuum. This allows the convenient exchange ofbuffers, if the buffer required for specific binding of bindingcompounds is different that the buffer required for either singletoxygen generation or separation. For example, in the case ofantibody-based binding compounds, a high salt buffer is required. Ifelectrophoretic separation of the released tags is employed, then betterperformance is achieved by exchanging the buffer for one that has alower salt concentration suitable for electrophoresis. In thisembodiment, instead of attaching a photosensitizer directly to a bindingcompound, such as an antibody, a cleaving probe comprises twocomponents: antibody (232) derivatized with a capture moiety, such asbiotin (indicated in FIG. 5C as “bio”) and photosensitizer bead (338)whose surface is derivatized with an agent (234) that specifically bindswith the capture moiety, such as avidin or streptavidin. Complexes (230)are then captured (236) by photosensitizer beads (338) by way of thecapture moiety, such as biotin. Conveniently, if the pore diameter ofthe filter membrane is selected so that photosensitizer beads (338)cannot pass, then a buffer exchange also serves to remove unboundbinding compounds, which leads to an improved signal. As illustrated inFIG. 5D, after an appropriate buffer for separation has been added, ifnecessary, photosensitizer beads (338) are illuminated (240) so thatsinglet oxygen is generated (242) and molecular tags are released (244).Such released molecular tags (346) are then separated to form separationprofile (352) and dimers are quantified ratiometrically from peaks (348)and (350). Photosensitizer beads may be used in either homogeneous orheterogeneous assay formats.

Preferably, when analytes, such as cell surface receptors, are beingdetected or antigen in a fixed sample, a cleaving probe may comprise aprimary haptenated antibody and a secondary anti-hapten binding proteinderivatized with multiple photosensitizer molecules. A preferred primaryhaptenated antibody is a biotinylated antibody, and preferred secondaryanti-hapten binding proteins may be either an anti-biotin antibody orstreptavidin. Other combinations of such primary and secondary reagentsare well known in the art, e.g. Haugland, Handbook of Fluorescent Probesand Research Reagents, Ninth Edition (Molecular Probes, Eugene, Oreg.,2002). An exemplary combination of such reagents is illustrated in FIG.5E. There binding compounds (366 and 368) having releasable tags (“mT₁”and “mT₂” in the Figure), and primary antibody (368) derivatized withbiotin (369) are specifically bound to different epitopes of receptordimer (362) in membrane (360). Biotin-specific binding protein (370),e.g. streptavidin, is attached to biotin (369) bringing multiplephotosensitizers (372) into effective proximity of binding compounds(366 and 368). Biotin-specific binding protein (370) may also be ananti-biotin antibody, and photosensitizers may be attached via freeamine group on the protein by conventional coupling chemistries, e.g.Hermanson (cited above). An exemplary photosensitizer for such use is anNHS ester of methylene blue prepared as disclosed in Shimadzu et al.,European patent publication 0510688.

E. Assay Conditions

The following general discussion of methods and specific conditions andmaterials are by way of illustration and not limitation. One of ordinaryskill in the art will understand how the methods described herein can beadapted to other applications, particularly with using differentsamples, cell types and target complexes.

In conducting the methods of the invention, a combination of the assaycomponents is made, including the sample being tested, the bindingcompounds, and optionally the cleaving probe. Generally, assaycomponents may be combined in any order. In certain applications,however, the order of addition may be relevant. For example, one maywish to monitor competitive binding, such as in a quantitative assay. Orone may wish to monitor the stability of an assembled complex. In suchapplications, reactions may be assembled in stages, and may requireincubations before the complete mixture has been assembled, or beforethe cleaving reaction is initiated.

The amounts of each reagent are usually determined empirically. Theamount of sample used in an assay will be determined by the predictednumber of target complexes present and the means of separation anddetection used to monitor the signal of the assay. In general, theamounts of the binding compounds and the cleaving probe are provided inmolar excess relative to the expected amount of the target molecules inthe sample, generally at a molar excess of at least 1.5, more desirablyabout 10-fold excess, or more. In specific applications, theconcentration used may be higher or lower, depending on the affinity ofthe binding agents and the expected number of target molecules presenton a single cell. Where one is determining the effect of a chemicalcompound on formation of oligomeric cell surface complexes, the compoundmay be added to the cells prior to, simultaneously with, or afteraddition of the probes, depending on the effect being monitored.

The assay mixture is combined and incubated under conditions thatprovide for binding of the probes to the cell surface molecules, usuallyin an aqueous medium, generally at a physiological pH (comparable to thepH at which the cells are cultures), maintained by a buffer at aconcentration in the range of about 10 to 200 mM. Conventional buffersmay be used, as well as other conventional additives as necessary, suchas salts, growth medium, stabilizers, etc. Physiological and constanttemperatures are normally employed. Incubation temperatures normallyrange from about 4° C. to 70° C., usually from about 15° C. to 45° C.,more usually 25° C. to 37° C.

After assembly of the assay mixture and incubation to allow the probesto bind to cell surface molecules, the mixture is treated to activatethe cleaving agent to cleave the tags from the binding compounds thatare within the effective proximity of the cleaving agent, releasing thecorresponding tag from the cell surface into solution. The nature ofthis treatment will depend on the mechanism of action of the cleavingagent. For example, where a photosensitizer is employed as the cleavingagent, activation of cleavage will comprise irradiation of the mixtureat the wavelength of light appropriate to the particular sensitizerused.

Following cleavage, the sample is then analyzed to determine theidentity of tags that have been released. Where an assay employing aplurality of binding compounds is employed, separation of the releasedtags will generally precede their detection. The methods for bothseparation and detection are determined in the process of designing thetags for the assay. A preferred mode of separation employselectrophoresis, in which the various tags are separated based on knowndifferences in their electrophoretic mobilities.

As mentioned above, in some embodiments, if the assay reactionconditions may interfere with the separation technique employed, it maybe necessary to remove, or exchange, the assay reaction buffer prior tocleavage and separation of the molecular tags. For example, assayconditions may include salt concentrations (e.g. required for specificbinding) that degrade separation performance when molecular tags areseparated on the basis of electrophoretic mobility. Thus, such high saltbuffers may be removed, e.g. prior to cleavage of molecular tags, andreplaced with another buffer suitable for electrophoretic separationthrough filtration, aspiration, dilution, or other means.

4. Disease Associated with Undesirable Angiogenesis

According to the present invention, multiple cellular components andtheir interactions in endothelial cells are analyzed to provideinformation for assessing angiogenesis and monitoring the diseasestatus.

As used herein, the term “disease status” includes, but is not limitedto, the following features: likelihood of contracting a disease,presence or absence of a disease, prognosis of disease severity, andlikelihood that a patient will respond to treatment by a particulartherapeutic agent that acts through a receptor complex. In regard tocancer, “disease status” further includes detection of precancerous orcancerous cells or tissues, the selection of patients that are likely torespond to treatment by a therapeutic agent that acts through inhibitionof angiogenesis, directly or indirectly.

Examples of diseases associated with undesirable angiogenesis include,but are not limited to, restenosis (e.g. coronary, carotid, and cerebrallesions), benign tumors, a various types of cancers such as primarytumors and tumor metastasis, hematological disorders, abnormalstimulation of endothelial cells (atherosclerosis), insults to bodytissue due to surgery, abnormal wound healing, abnormal angiogenesis,diseases that produce fibrosis of tissue, repetitive motion disorders,disorders of tissues that are not highly vascularized, and proliferativeresponses associated with organ transplants.

Generally, cells in a benign tumor retain their differentiated featuresand do not divide in a completely uncontrolled manner. A benign tumor isusually localized and nonmetastatic. Specific types benign tumorsinclude, but are not limited to, hemangiomas, hepatocellular adenoma,cavernous haemangioma, focal nodular hyperplasia, acoustic neuromas,neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma, lipomas,leiomyomas, mesotheliomas, teratomas, myxomas, nodular regenerativehyperplasia, trachomas and pyogenic granulomas.

In a malignant tumor cells become undifferentiated, do not respond tothe body's growth control signals, and multiply in an uncontrolledmanner. The malignant tumor is invasive and capable of spreading todistant sites (metastasizing). Malignant tumors are generally dividedinto two categories: primary and secondary. Primary tumors arisedirectly from the tissue in which they are found. A secondary tumor, ormetastasis, is a tumor which originated elsewhere in the body but hasnow spread to a distant organ. The common routes for metastasis aredirect growth into adjacent structures, spread through the vascular orlymphatic systems, and tracking along tissue planes and body spaces(peritoneal fluid, cerebrospinal fluid, etc.

Specific types of cancers or malignant tumors, either primary orsecondary, include, but are not limited to, breast cancer, skin cancer,bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer,cancer of the larynx, gallbladder, pancreas, rectum, parathyroid,thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi,kidneys, basal cell carcinoma, squamous cell carcinoma of bothulcerating and papillary type, metastatic skin carcinoma, osteo sarcoma,Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor,small-cell lung tumor, islet cell tumor, primary brain tumor, acute andchronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuromas,intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoidhabitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyoma tumor,cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma,soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosisfungoides, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and othersarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera,adenocarcinoma, glioblastoma multiforma, medulloblastoma, leukemias,lymphomas, malignant melanomas, epidermoid carcinomas, and othercarcinomas and sarcomas.

Examples of the hematological disorder include, but are not limited to,acute myeloid leukemia, acute promyelocytic leukemia, acutelymphoblastic leukemia, chronic myelogenous leukemia, themyelodysplastic syndromes, and sickle cell anemia. The proliferativeresponses associated with organ transplantation include thoseproliferative responses contributing to potential organ rejections orassociated complications. Specifically, these proliferative responsesmay occur during transplantation of the heart, lung, liver, kidney, andother body organs or organ systems.

Abnormal angiogenesis includes those abnormal angiogenesis accompanyingrheumatoid arthritis, ischemic-reperfusion related brain edema andinjury, cortical ischemia, ovarian hyperplasia and hypervascularity,(polycystic ovary syndrom), endometriosis, psoriasis, diabeticretinopaphy, and other ocular angiogenic diseases such as retinopathy ofprematurity (retrolental fibroplastic), macular degeneration, cornealgraft rejection, neuroscular glaucoma and Oster Webber syndrome.

Diseases associated with abnormal angiogenesis require or inducevascular growth. For example, corneal angiogenesis involves threephases: a pre-vascular latent period, active neovascularization, andvascular maturation and regression. The identity and mechanism ofvarious angiogenic factors, including elements of the inflammatoryresponse, such as leukocytes, platelets, cytokines, and eicosanoids, orunidentified plasma constituents have yet to be revealed.

Specific types of diseases associated with abnormal angiogenesis includeretinaVchoroidal neuvascularization and corneal neovascularization.Examples of retinal/choroidal neuvascularization include, but are notlimited to, Bests diseases, myopia, optic pits, Stargarts diseases,Pagets disease, vein occlusion, artery occlusion, sickle cell anemia,sarcoid, syphilis, pseudoxanthoma elasticum carotid obstructivediseases, chronic uveitis/vitritis, mycobacterial infections, Lyme'sdisease, systemic lupus erythematosis, retinopathy of prematurity, Ealesdisease, diabetic retinopathy, macular degeneration, Bechets diseases,infections causing a retinitis or chroiditis, presumed ocularhistoplasmosis, pars planitis, chronic retinal detachment,hyperviscosity syndromes, toxoplasmosis, trauma and post-lasercomplications, diseases associated with rubesis (neovascularization ofthe angle) and diseases caused by the abnormal proliferation offibrovascular or fibrous tissue including all forms of proliferativevitreoretinopathy. Examples of corneal neuvascularization include, butare not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency,contact lens overwear, atopic keratitis, superior limbic keratitis,pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis,diabetic retinopathy, retinopathy of prematurity, corneal graftrejection, Mooren ulcer, Terrien's marginal degeneration, marginalkeratolysis, polyarteritis, Wegener sarcoidosis, Scleritis, periphigoidradial keratotomy, neovascular glaucoma and retrolental fibroplasia,syphilis, Mycobacteria infections, lipid degeneration, chemical burns,bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpeszoster infections, protozoan infections and Kaposi sarcoma.

Examples of chronic inflammatory disease include, but are not limitedto, inflammatory bowel diseases such as Crohn's disease and ulcerativecolitis, psoriasis, sarcoidois, and rheumatoid arthritis.

Inflammatory bowel diseases such as Crohn's disease and ulcerativecolitis are characterized by chronic inflammation and angiogenesis atvarious sites in the gastrointestinal tract. For example, Crohn'sdisease occurs as a chronic transmural inflammatory disease that mostcommonly affects the distal ileum and colon but may also occur in anypart of the gastrointestinal tract from the mouth to the anus andperianal area. Patients with Crohn's disease generally have chronicdiarrhea associated with abdominal pain, fever, anorexia, weight lossand abdominal swelling. Ulcerative colitis is also a chronic,nonspecific, inflammatory and ulcerative disease arising in the colonicmucosa and is characterized by the presence of bloody diarrhea. Theseinflammatory bowel diseases are generally caused by chronicgranulomatous inflammation throughout the gastrointestinal tract,involving new capillary sprouts surrounded by a cylinder of inflammatorycells.

The inflammatory bowel diseases also exhibit extra intestinalmanifestations, such as skin lesions. Such lesions are characterized byinflammation and abnormal angiogenesis and can occur at many sites otherthe gastrointestinal tract. Inhibition of angiogenesis by thecombination of a camptothecin compound and gemcitabine should reduce theinflux of inflammatory cells and prevent the lesion formation.

Sarcoidois, another chronic inflammatory disease, is characterized as amultisystem granulomatous disorder. The granulomas of this disease canform anywhere in the body and, thus, the symptoms depend on the site ofthe granulomas and whether the disease is active. The granulomas arecreated by the angiogenic capillary sprouts providing a constant supplyof inflammatory cells. By using a combination of a camptothecin compoundand gemcitabine to inhibit angiogenesis, such granulomas formation canbe inhibited. Psoriasis, also a chronic and recurrent inflammatorydisease, is characterized by papules and plaques of various sizes.

Rheumatoid arthritis (RA) is also a chronic inflammatory diseasecharacterized by non-specific inflammation of the peripheral joints. Itis believed that the blood vessels in the synovial lining of the jointsundergo angiogenesis. In addition to forming new vascular networks, theendothelial cells release factors and reactive oxygen radicals that leadto pannus growth and cartilage destruction. The factors involved inangiogenesis may actively contribute to, and help maintain, thechronically inflamed state of rheumatoid arthritis.

5. Examples of Antiangiogenic Agents

According to the present invention, multiple cellular components andtheir interactions in endothelial cells are analyzed to provideinformation for assessing angiogenesis and monitoring the disease statuswhich includes an individual's response or resistance to the treatmentof an antiangiogenic agent.

A wide variety of anti-angiogenic agents have been developed. Examplesof anti-angiogenesis agents include, but are not limited to, retinoidacid and derivatives thereof, 2-methoxyestradiol, ANGIOSTATIN™ protein,ENDOSTATIN™ protein, suramin, squalamine, tissue inhibitor ofmetalloproteinase-I, tissue inhibitor of metalloproteinase-2,plasminogen activator inhibitor-1, plasminogen activator inhibitor-2,cartilage-derived inhibitor, paclitaxel, platelet factor 4, protaminesulphate (clupeine), sulphated chitin derivatives (prepared from queencrab shells), sulphated polysaccharide peptidoglycan complex (sp-pg),staurosporine, modulators of matrix metabolism, including for example,proline analogs ((l-azetidine-2-carboxylic acid (LACA),cishydroxyproline, d,l-3,4-dehydroproline, thiaproline], α,α-dipyridyl,.beta.-aminopropionitrile fumarate,4-propyl-5-(4-pyridinyl)-2(3h)-oxazolone; methotrexate, mitoxantrone,heparin, interferons, 2 macroglobulin-serum, chimp-3, chymostatin,∃-cyclodextrin tetradecasulfate, eponemycin; fumagillin, gold sodiumthiomalate, d-penicillamine (CDPT), ∃-1-anticollagenase-serum,α2-antiplasmin, bisantrene, lobenzarit disodium,n-(2-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”,thalidomide; angostatic steroid, cargboxynaminolmidazole;metalloproteinase inhibitors such as BB94. Other anti-angiogenesisagents include antibodies, preferably monoclonal antibodies againstthese angiogenic growth factors: bFGF, aFGF, FGF-5, VEGF isoforms,VEGF-C, HGF/SF and Ang-1/Ang-2. Ferrara N. and Alitalo, K. “Clinicalapplication of angiogenic growth factors and their inhibitors” (1999)Nature Medicine 5:1359-1364.

Mechanistically, according to Kerbel and Folkman (2002) (Nature Rev.2:727-739) there are two classes of antiangiogenic agents: direct andindirect angiogenesis inhibitors. Examples of direct angiogenesisinhibitors that are undergoing clinical trials are listed in Table 1;and those of indirect angiogenesis inhibitors in Table 2.

Direct angiogenesis inhibitors, such as vitaxin, angiostatin and others,prevent vascular endothelial cells from proliferating, migrating oravoiding cell death in response to a spectrum of pro-angiogenicproteins, including VEGF, bFGF, IL-8, platelet-derived growth factor(PDGF) and PD-EGF (Table 1). Direct angiogenesis inhibitors are theleast likely to induce acquired drug resistance, because they targetgenetically stable endothelial cells rather than unstable mutating tumorcells. Kerbel (1991) Bioessays 13:31-36. Tumors that are treated withdirect-acting anti-angiogenic therapy did not develop drug resistance inmice (Boehm et al. (1997) Nature 390:404-407).

Indirect angiogenesis inhibitors generally prevent the expression of orblock the activity of a tumor protein that activates angiogenesis, orblock the expression of its receptor on endothelial cells (Table 2).Many of these tumor-cell proteins are the products of oncogenes thatdrive the angiogenic switch. The activities of oncogene andtumor-suppressor gene products were initially studied in in vitro assaysthat monitored cancer-cell proliferation, apoptosis resistance,immortalization and anchorage independence. Kerbel et al. (1998) Mol.Med. 4: 286-296. Because the increased cancer-cell proliferation anddecreased apoptosis that was associated with oncogene activation invitro correlated so well with tumor growth in vivo, there was no reasonto suspect that these new anticancer drugs (for example,signal-transduction inhibitors such as trastuzumab (HERCEPTIN)) couldalso block the angiogenic output of a tumor. But activating mutations inoncogenes, as well as in the anti-apoptotic factor BCL2, have been shownto cause tumor cells to upregulate angiogenic proteins and todownregulate inhibitors of angiogenesis. Fernandez et al. (2001) J.Natl. Cancer Inst. 93:33-38.

TABLE 1 Drug Endothelia-cell target Reference(s) Angiostatin Binds toATP synthase, angiomotin Troyanovsky et al. and annexin II onendothelial cells (2001) J. Cell to inhibit endothelial-cell Biol. 152:1247- proliferation and migration 1254 Bevacizumab Recombinant humanizedBevacizumab (2002) (Avastin) monoclonal antibody against Drugs R. D. 3:vascular endothelial growth factor 28-30 (VEGF) Arresten Believed tobind integrin-α₁β₁ to Colorado et al. inhibit endothelial-cell (2000)Cancer proliferation, migration, tube Res. 60: 2520- formation andneovascularization 2526 Canstatin Believed to bind integrin-α_(v)β₃ toKamphaus et al. inhibit endothelial-cell (2000) J. Biol. proliferation,migration and tube Chem. 275: 1209- formation 1215 Combreta-Microtubules: induces Kanthou et al. statin reorganization of the actin(2002) Blood cytoskeleton and early membrane 99: 2060-2069 blebbing inhuman endothelial cells Endostatin Believed to target integrin-α₅β₁ toDixelius et al. inhibit endothelial-cell proliferation (2000) Blood 95:and migration, and induce 3403-3411 apoptosis of proliferating O'Reillyet al. endothelial cells (R. Kalluri, (1997) Cell 88: personalcommunication); 277-285 endostatin does not affect wound healing NM-3 Anisocoumarin small-molecule Reimer et al. inhibitor of VEGF. It was shownto (2002) Cancer selective{circumflex over ( )} inhibit endothelial-cellRes. 62: 789-795 proliferation, sprouting and tube formation in vitroThrombo- Blocks endothelial-cell migration Dameron et al. spondin andneovascularization in the (1994) cornea, but might not be specificScience 265: for endothelial cells 1582-1584. Tumstatin Binds tointegrin α_(v)β₃ endothelial Maeshima et al. cells; inhibitsendothelial-cell (2001) J. Biol. proliferation and Chem. 276: 15240-neovascularization 15248. Maeshima et al. (2002) Science 295: 140-1432-methoxy- Inhibits micro-tubule function in D'Amato et estradiolproliferating endothelial cells, al. (1994) Proc. resulting inendothelial-cell Natl. Acad. Sci. apoptosis 91: 4082-4085 Vitaxin Ahumanized monoclonal antibody Gutheil et al. against integrin α_(v)β₃(2002) Clin. Cancer Res. 6: 3056-3061

TABLE 2 Cancer-cell Pro-angiogenic target proteins Drug Reference(s) EGFVEGF; bFGF; ZD1839 (Iressa); Ciardiello receptor TGF-α ZD6474; OSI774 etal. (2001) tyrosine (Tarceva); CI1033; Clin. Cancer kinase PKI1666;IMC225 Res. 7: 1459- (Erbitux) 1465. VEGF VEGF PTK787; ZD6474; Tille tal. (2001) receptor receptor on SU6668; SU11248; J. Pharmacol.endothelium Semaxanib (SU5416); Exp. Ther. 299: IMC-1C11 1073-1086.Hoekman (2001) Cancer J. 7: S134-S138. PDGF PDGF PTK787; Tille t al.(2001) receptor receptor SU11248; J. Pharmacol. SU6668 Exp. Ther. 299:1073-1086. ERBB- VEGF, angio- Herceptin Kerbel et al. 2(HER-2/neupoietin-1, (1998) Mol. Med. receptor TGF-β, PAI1; 4: 286-296. tyrosineupregulates Izumi (2002) kinase) thrombo- Nature spondin-1 416: 279-280.Interferon Inhibits IFN-α Singh et al. (iFN)-α expression of (1996)Proc. receptor bFGF by Natl. Acad. Sci. cancer cells 92: 4562-4566. FGFFGF SU11248; Tille t al. receptor Receptor SU6668 (2001) J. Pharmacol.Exp. Ther. 299: 1073-1086.

In the clinic, antiangiogenic drugs are often combined with othertherapeutic agents to achieve maximum therapeutic efficacy and/or toreduce drug toxicity, especially in the treatment of heterogenousdiseases such as cancer.

Examples of such therapeutic agents that can be combined withantiangiogenic agents include, but are not limited to, alkylatingagents, antibiotic agents, antimetabolic agents, hormonal agents,plant-derived agents, and biologic agents.

The alkylating agents are polyfunctional compounds that have the abilityto substitute alkyl groups for hydrogen ions. Examples of alkylatingagents include, but are not limited to, bischloroethylamines (nitrogenmustards, e.g. chlorambucil, cyclophosphamide, ifosfamide,mechlorethamine, melphalan, uracil mustard), aziridines (e.g. thiotepa),alkyl alkone sulfonates (e.g. busulfan), nitrosoureas (e.g. carmustine,lomustine, streptozocin), nonclassic alkylating agents (altretamine,dacarbazine, and procarbazine), platinum compounds (carboplastin andcisplatin). These compounds react with phosphate, amino, hydroxyl,sulfihydryl, carboxyl, and imidazole groups. Under physiologicalconditions, these drugs ionize and produce positively charged ion thatattach to susceptible nucleic acids and proteins, leading to cell cyclearrest and/or cell death.

The antibiotic agents are a group of drugs that are produced in a mannersimilar to antibiotics as a modification of natural products. Examplesof antibiotic agents include, but are not limited to, anthracyclines(e.g. doxorubicin, daunorubicin, epirubicin, idarubicin andanthracenedione), mitomycin C, bleomycin, dactinomycin, plicatomycin.These antibiotic agents interfere with cell growth by targetingdifferent cellular components. For example, anthracyclines are generallybelieved to interfere with the action of DNA topoisomerase II in theregions of transcriptionally active DNA, which leads to DNA strandscissions. Bleomycin is generally believed to chelate iron and forms anactivated complex, which then binds to bases of DNA, causing strandscissions and cell death.

The antimetabolic agents are a group of drugs that interfere withmetabolic processes vital to the physiology and proliferation of cancercells. Actively proliferating cancer cells require continuous synthesisof large quantities of nucleic acids, proteins, lipids, and other vitalcellular constituents. Many of the antimetabolites inhibit the synthesisof purine or pyrimidine nucleosides or inhibit the enzymes of DNAreplication. Some antimetabolites also interfere with the synthesis ofribonucleosides and RNA and/or amino acid metabolism and proteinsynthesis as well. By interfering with the synthesis of vital cellularconstituents, antimetabolites can delay or arrest the growth of cancercells. Examples of antimetabolic agents include, but are not limited to,fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate, leucovorin,hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine,pentostatin, fludarabine phosphate, cladribine (2-CDA), andasparaginase.

The hormonal agents are a group of drug that regulate the growth anddevelopment of their target organs. Most of the hormonal agents are sexsteroids and their derivatives and analogs thereof, such as estrogens,androgens, and progestins. These hormonal agents may serve asantagonists of receptors for the sex steroids to down regulate receptorexpression and transcription of vital genes. Examples of such hormonalagents are synthetic estrogens (e.g. diethylstibestrol), antiestrogens(e.g. tamoxi fen, toremifene, fluoxymesterol and raloxifene),antiandrogens (bicalutamide, nilutamide, flutamide), aromataseinhibitors (e.g., aminoglutethimide, anastrozole and tetrazole),ketoconazole, goserelin acetate, leuprolide, megestrol acetate andmifepristone.

Plant-derived agents are a group of drugs that are derived from plantsor modified based on the molecular structure of the agents. Examples ofplant-derived agents include, but are not limited to, vinca alkaloids(e.g., vincristine, vinblastine, vindesine, vinzolidine andvinorelbine), podophyllotoxins (e.g., etoposide (VP-16) and teniposide(VM-26)), taxanes (e.g., paclitaxel and docetaxel). These plant-derivedagents generally act as antimitotic agents that bind to tubulin andinhibit mitosis. Podophyllotoxins such as etoposide are believed tointerfere with DNA synthesis by interacting with topoisomerase II,leading to DNA strand scission. Biologic agents are a group ofbiomolecules that elicit cancer/tumor regression when used alone or incombination with chemotherapy and/or radiotherapy.

Examples of biologic agents include, but are not limited to,immuno-modulating proteins such as cytokines, monoclonal antibodiesagainst tumor antigens, tumor suppressor genes, and cancer vaccines.Combination therapy including a camptothecin compound, gemcitabine andthe biologic agent may have therapeutic synergistic effects on cancer,enhance the patient's immune responses to tumorigenic signals, andreduce potential sides affects associated with this chemotherapeuticagent.

Cytokines possess profound immunomodulatory activity. Some cytokinessuch as interleukin-2 (IL-2, aldesleukin) and interferon α (IFN-α)demonstrated antitumor activity and have been approved for the treatmentof patients with metastatic renal cell carcinoma and metastaticmalignant melanoma. IL-2 is a T-cell growth factor that is central toT-cell-mediated immune responses. The selective antitumor effects ofIL-2 on some patients are believed to be the result of a cell-mediatedimmune response that discriminate between self and nonself. Examples ofinterleukins include, but are not limited to, interleukin 2 (IL-2), andinterleukin 4 (IL-4), interleukin 12 (IL-12).

Interferon α include more than 23 related subtypes with overlappingactivities, all of the IFN-α subtypes within the scope of the presentinvention. IFN-α has demonstrated activity against many solid andhematologic malignancies, the later appearing to be particularlysensitive. Examples of interferons include, but are not limited to,interferon a interferon ∃ (fibroblast interferon) and interferon γ(fibroblast interferon).

Other cytokines include those cytokines that exert profound effects onhematopoiesis and immune functions. Examples of such cytokines include,but are not limited to erythropoietin (epoietin α), granulocyte-CSF(filgrastin), and granulocyte, macrophage-CSF (sargramostim).

Other immuno-modulating agents other than cytokines may also be used inconjunction with CPT and a COX-2 inhibitor to inhibit abnormal cellgrowth. Examples of such immuno-modulating agents include, but are notlimited to bacillus Calmette-Guerin, levamisole, and octreotide, along-acting octapeptide that mimics the effects of the naturallyoccurring hormone somatostatin.

An example of monoclonal antibodies against tumor antigens is RITUXAN®(Rituximab) that is raised against CD20 on lymphoma cells andselectively deplete normal and malignant CD20+ pre-B and mature B cells.RITUXAN® is used as single agent for the treatment of patients withrelapsed or refractory low-grade or follicular, CD20+, B cellnon-Hodgkin's lymphoma.

Other examples of anti-cancer antibodies include, but are not limitedto, MYLOTARG® (gemtuzumab ozogamicin) which is an monoclonal antibodyapproved for treating acute myeloid leukemia (AML), CAMPATH®(alemtuzumab) for B cell chronic lymphocytic leukemia, ZEVALIN®(ibritumomab yiuxetan) for non-Hodgkin's lymphoma (NHL), PANOREX®(edrecolomab) for colorectal cancer, BEXXAR® (tositumomab) for treatingNHL, ERBITUX® (cetuximab) which is a monoclonal antibody targetingepidermal growth factor (EGF) and for treating various cancers, andpemtumomab for treating ovarian cancer.

Tumor suppressor genes are genes that function to inhibit the cellgrowth and division cycles, thus preventing the development ofneoplasia. Mutions in tumor suppressor genes cause the cell to ignoreone or more of the components of the network of inhibitory signals,overcoming the cell cycle check points and resulting in a higher rate ofcontrolled cell growth—cancer. Examples of the tumor suppressor genesinclude, but are not limited to, DPC-4, NF-1, NF-2, RB, p53, WT1, BRCA1and BRCA2.

DPC-4 is involved in pancreatic cancer and participates in a cytoplasmicpathway that inhibits cell division. NF-1 codes for a protein thatinhibits Ras, a cytoplasmic inhibitory protein. NF-1 is involved inneurofibroma and pheochromocytomas of the nervous system and myeloidleukemia. NF-2 encodes a nuclear protein that is involved in meningioma,schwanoma, and ependymoma of the nervous system. RB codes for the pRBprotein, a nuclear protein that is a major inhibitor of cell cycle. RBis involved in retinoblastoma as well as bone, bladder, small cell lungand breast cancer. P53 codes for p53 protein that regulates celldivision and can induce apoptosis. Mutation and/or inaction of p53 arefound in a wide ranges of cancers. WT1 is involved in Wilms tumor of thekidneys. BRCA1 is involved in breast and ovarian cancer, and BRCA2 isinvolved in breast cancer. The tumor suppressor gene can be transferredinto the tumor cells where it exerts its tumor suppressing functions.

Cancer vaccines are a group of agents that induce the body's specificimmune response to tumors. Most of cancer vaccines under research anddevelopment and clinical trials are tumor-associated antigens (TAAs).TAA are structures (i.e. proteins, enzymes or carbohydrates) which arepresent on tumor cells and relatively absent or diminished on normalcells. By virtue of being fairly unique to teh tumor cell, TAAs providetargets for the immune system to recognize and cause their destruction.Example of TAAs include, but are not limited to gangliosides (GM2),prostate specific antigen (PSA), α-fetoprotein (AFP), carcinoembryonicantigen (CEA) (produced by colon cancers and other adenocarcinomas, e.g.breast, lung, gastric, and pancreas cancer s), melanoma associatedantigens (MART-1, gp100, MAGE 1,3 tyrosinase), papillomavirus E6 and E7fragments, whole cells or portions/lysates of autologous tumor cells andallogeneic tumor cells.

An adjuvant may be used to augment the immune response to TAAs. Examplesof adjuvants include, but are not limited to, bacillus Calmette-Guerin(BCG), endotoxin lipopolysaccharides, keyhole limpet hemocyanin (GKLH),interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor(GM-CSF) and cytoxan, a chemotherapeutic agent which is believe toreduce tumor-induced suppression when given in low doses.

The following example serves to more fully describe the manner of usingthe above-described invention. It is understood that the example in noway serves to limit the scope of this invention, but rather is presentedfor illustrative purpose. All references cited herein are incorporatedby reference in their entirety.

EXAMPLES Sources of Materials Used in Examples

Antibodies specific for receptors, adaptor molecules, and normalizationstandards are obtained from commercial vendors, including Labvision,Cell Signaling Technology, and BD Biosciences. All cell lines werepurchased from ATCC or Cambrex Bio Science Walkersvile, Inc.(Walkersville, Md.). All human snap-frozen tissue samples were purchasedfrom either William Bainbridge Genome Foundation (Seattle, Wash.) or BioResearch Support (Boca Raton, Fla.) and were approved by InstitutionalResearch Board (IRB) at the supplier.

The molecular tag-antibody conjugates used below are formed by reactingNHS esters of the molecular tag with a free amine on the indicatedantibody using conventional procedures. Molecular tags, identified belowby their “Pro_N” designations, are either disclosed in the followingreferences: Singh et al., U.S. patent publications, 2003/017915 and2002/0013126, all of which are incorporated by reference, or theirformulas are shown in FIGS. 11A and 11B. The latter molecular tags aresynthesized and attached to binding compounds as taught in the two abovereferences. Briefly, binding compounds below are moleculartag-monoclonal antibody conjugates formed by reacting an NHS ester of amolecular tag with free amines of the antibodies in a conventionalreaction.

Example 1 Immunomagnetic Isolation of Human Endothelial Cells

Substantially pure CEC and CECP can be isolated by using theimmunomagnetic isolation/enrichment technique described in Kinzler etal. (2000) Science 289:1197-1202. Briefly, the epithelial andhematopoietic cell fractions in the peripheral blood samples aresequentially removed via negative selection via antibody-linked magneticbeads (BerEP4 beads—Epithelial, CD45 beads—Pan leukocyte, CD64beads—Macrophages, and CD14 beads—Monocytes). The remaining cells arestained with P1H12 antibodies and are isolated via positive selectionwith magnetic beads.

In this example, CEC and CECP were isolated by using a protocol modifiedfrom Voest et al. (2004) Annals of Oncology 15: 139-145 as follows:

-   -   1) Magnetic beads (Dynal M450 IgG_(1,) Dynal AS, Oslo, Norway)        were conjugated to a monoclonal antibody for CD146, MCAM, an        endothelial cell surface junction protein.    -   2) Whole blood was diluted 1:3 with 0.9% NaCl and incubated for        30 min with 20 uL (2.8×10⁶) antibody-coupled magnetic beads at        4° C. on a roller bench.    -   3) Unbound cells were removed by magnetic separation using a        MPC-L magnet (Dynal, AS)    -   4) The beads-bound cell fraction was rinsed with        phosphate-buffered saline-bovine serum albumin (PBS-BSA 0.1%).        Cells are now ready for lysis.

Human umbilical vein endothelial cells (HUVEC) were isolate from U937 byusing CD146 conjugated magnetic beads following the protocols listedbelow.

Isolation of HUVEC from U937 Using CD146 Conjugated Magnetic Beads

-   I. Wash Beads    -   1) Vortex to resuspend beads in storage buffer.    -   2) Pipette 350uL beads (30 mg/mL) to 1.5 mL Tube.    -   3) Capture beads with magnet for 60 seconds. Remove supernatant.    -   4) Remove tube from magnet. Add 1.5 mL 0.1% BSA in PBS (pH 7.4).    -   5) Vortex to resuspend beads.    -   6) Capture beads with magnet for 60 seconds. Remove supernatant.    -   7) Repeat Steps 4-6. (Wash 1 more time with BSA/PBS).    -   8) Resuspend beads in 350 uL 0.1% BSA in PBS (pH 7.4).    -   9) Beads are 30 mg/mL final concentration.-   II. Incubation of Sample    -   1) Add 25 uL beads to each cel mixture (1 mL cells/sample)    -   2) Incubate the sample for 30 min @ 4° C. with gentle rotation.    -   3) Add 500 uL PBS +0.1% BSA    -   4) Capture beads with magnet for 2 min. Remove supernatant.    -   5) Remove tube from magnet. Add 1.5 mL 0.1% BSA in PBS (pH 7.4).    -   6) Vortex to resuspend beads.    -   7) Capture beads with magnet for 2 min. Remove supernatant.    -   8) Repeat Steps 5-7. (Wash 3 more times with BSA/PBS).    -   9) After the 4th wash resuspend the beads in 100 uL freshly made        Lysis Buffer.    -   10) Incubate on ice for 30 min.    -   11) Spin cells in microfuge at max rpm for 10 min.    -   12) Remove supernatant to new tube for assay or storage at −70°        C.

Example 2 Lysis of CEC or CECP

The isolated CEC or CECP were lysed according to the following protocol:

-   Protocol    -   Step        -   1) Seed 2 15 cm dishes with 6×10̂6 cells in 20 mL EGM        -   2) Prepare fresh lysis buffer        -   3) Starting now, always work on ice. Aspirate the medium.        -   4) Add 600 ul of fresh lysis buffer.        -   5) Swirl the plates to distribute the buffer evenly.        -   6) Scrap the cells.        -   7) Collect the crude lysate in 1.5-ml tube.        -   8) Wait for 30 min.        -   9) Spin down at 4 C at 14000 rpm for 10 min        -   10) Collect the supernatant in 1.5-ml new tube.        -   11) Store at −80 C for later use.-   The lysis buffer was prepared by mixing the following reagents:

Lysis Stock 10 ml buffer Reagents con. Final conc Vol 1) 10% tritonX-100 10 1 1.00 2) 1M Tris pH 7.5 1 0.05 0.50 3) 1M NaF 1 0.05 0.50 4)5M NaCl 5 0.1 0.20 5) 2M B-Glycerol- 1 0.05 0.50 phosphate 6) 0.1MNa3VO4 0.1 0.001 0.10 7) 1 mg/ml pepstatin 1 0.01 0.10 8) Complete mini1 tablet protease 9) 0.5M EDTA 0.5 0.005 0.10 Total (ml) 3.00 Water (ml)7.00

Example 3 Analysis of Cell Lysate for Protein Complexes of AngiogenicReceptors and Phosphorylation of Downstream Effector Proteins

Protein complexes formed by angiogenic receptors (e.g., VEGFR, Tie),such as the homodimers and heterodimers formed by VEGFR, were measuredin cell lysates from CEC, CECP, and cell lines such as human umbilicalvein endothelial cells (HUVEC), and human vulval carcinoma cell line(A431). Measurements were made using three binding compounds and acleaving probe by using the following protocol.

-   1) Add Lysis Buffer and Lysate into 96-well PCR plate to 30 uL final    volume.-   2) Add 5 uL antibody mix to each well.-   3) Incubate reactions at RT for 2 hours on plate shaker.-   4) Add 5 uL steptavidin-scissor mix to each well in dark room.    -   Mix is 32.5×. Added 15 uL to 472.5 uL Dilution Buffer (1% BSA in        PBS)-   5) Incubate reactions at RT for 45 minutes on plate shaker in dark    room.-   6) Pre-wet 96-well 2 micron membrane filter plates with 100 uL    Exchange Buffer I-   7) Drain filter plate and add the 40 uL reactions in the dark room.-   8) Drain the plate and add 150 uL Exchange Buffer I (1×PBS, 0.5%    Triton 1000) per well.-   9) Drain the plate and add 150 uL Exchange Buffer II (0.005 PBS) per    well. Repeat this step one more time.-   10) Drain the plate and add 30 uL Illumination Buffer (0.005 PBS)    w/CE standard to each well.-   11) Illuminate for 15 min at RT.-   12) Transfer the entire reaction to clean 96-well PCR plate.-   13) Aliquot 9 uL to CE plate and run.

Example 4 Analysis of Cell Lysates for VEGFR2 Homodimerization andReceptor Phosphorylation

In this example, VEGFR2-VEGFR2 homodimers and phosphorylation stateswere measured in cell lysates from several cell lines after treatmentwith various concentrations of vascular endothelial growth factor(VEGF). Measurements were made using three binding compounds and acleaving probe as described below.

Sample Preparation:

-   -   1. Serum-starve human umbilical vein endothelial cell line        (HUVEC) culture overnight before use.    -   2. Stimulate cell lines with VEGF in culture media for a set        period of time (1, 3, 5, 9, 14 minutes) at 37° C. Exemplary        doses of VEGF are 0, 2, 10, 25, 50, 250, 1000 ng/mL.    -   3. Aspirate culture media, transfer onto ice, and add lysis        buffer to lyse cells in situ.    -   4. Scrape and transfer lysate to microfuge tube. Incubate on ice        for 30 min. Microfuge at 14,000 rpm, 4° C., for 10 min.        (Centrifugation is optional.)    -   5. Collect supernatants as lysates and aliquot for storage at        −80° C. until use.

Assay:

-   Assay design: As illustrated diagrammatically in FIG. 7A,    VEGFR2-VEGFR2 (R2-R2) homodimers (900) are quantified    ratiometrically based on the binding of a cleaving probe (902), a    first bind compound (904) which is specific for the same antigenic    determinant on VEGFR2 as the cleaving probe, a second binding    compound (906) which is specific for a different antigenic    determinant on VEGFR2 from the cleaving probe, and a third binding    compound (908) which is specific for phosphorylated tyrosine. A    photosensitizer is attached to the cleaving probe (902) via an    avidin-biotin linkage, and the first, second, and third binding    compounds are labeled with molecular tags Pro10, Pro14, and Pro2,    respectively.-   The total assay volume was 40 ul. The lysate volume was adjusted to    30 ul with lysis buffer. The antibodies are diluted in lysis buffer    up to 10 ul. Typically ˜5000 to15000 cell-equivalent of lysates is    used per reaction. The detection limit is ˜1000 cell-equivalent of    lysates.-   Procedure: Final concentrations of pre-mixed binding compounds (i.e.    molecular tag- or biotin-antibody conjugates) in reaction:

Pro10_anti-VEGFR2: 0.25 ug/ml (VEGFR2 homodimer)

Pro14_anti-VEGFR2: 0.25 ug/ml (total VEGFR2)

Pro2_anti-phospho-Tyr: 0.0125 ug/ml (phosphorylation)

Biotin_anti-VEGFR2: 0.25 ug/ml

-   -   1. To assay 96-well, add 10 ul antibody mix to 30 ul lysate and        incubate for 1 hour at RT.    -   2. Add 2 ul streptavidin-derivatized cleaving probe (final 2        ug/well) to assay well and incubate for 45 min.    -   3. Add 150 ul of wash buffer to 96-well filter plate (Millipore        MAGVN2250) and incubate for 1 hr at RT for blocking.    -   4. Empty filter plate by vacuum suction. Transfer assay        reactions to filter plate and apply vacuum to empty.    -   5. Add 200 ul wash buffer and apply vacuum to empty. Repeat one        time.    -   6. Add 200 ul illumination buffer and apply vacuum to empty.        Repeat one time.    -   7. Add 30 ul illumination buffer and illuminate for 20 min.    -   8. Transfer 10 ul of each reaction to CE assay plate for        analysis using an ABI3100 CE instrument with a 22 cm capillary        (injection conditions: 5 kV, 75 sec, 30° C.; run conditions: 425        sec, 30° C.).

-   Assay buffers are as follows:

-   Lysis Buffer (made fresh and stored on ice)

Final ul Stock 1% Triton X-100 1000 10% 20 mM Tris-HCl (pH 7.5) 200  1M100 mM NaCl 200  5M 50 mM NaF 500  1M 50 mM Na beta-glycerophosphate1000 0.5M 1 mM Na₃VO₄ 100 0.1M 5 mM EDTA 100 0.5M 10 ug/ml pepstatin 1001 mg/ml 1 tablet (per 10 ml) Roche Complete N/A N/A protease inhibitor(#1836170) Water 6500 N/A 10 ml Total

-   Wash buffer (stored at 4° C.)

Final ml Stock 1% NP-40 50 10% 1x PBS 50 10x 150 mM NaCl 15   5M 5 mMEDTA 5 0.5M Water 380 N/A 500 ml Total

-   Illumination buffer:

Final ul Stock 0.005x PBS 50  1x CE std 3 100x 10 mM Tris-HCl (pH 8.0)0.1M 10 pM A160 1 nM 10 pM A315 1 nM 10 pM HABA 1 nM Water 10,000 N/A 10ml Total

Data Analysis:

-   -   1. Normalize relative fluorescence units (RFU) signal of each        molecular tag against CE reference standard A315 (a        fluorescein-derivatized deoxyadenosine monophosphate that has        known peak position relative to molecular tags from the assay        upon electrophoretic separation).    -   2. Subtract RFU of “no lysate” background control from        corresponding molecular tag signals.    -   3. Report homodimerization of VEGFR2 as the corresponding RFU        ratiometric to RFU from Pro14_anti-VEGFR2 from assay wells using        biotin_anti-VEGFR2.    -   4. Report receptor phosphorylation for VEGFR2 as RFU from        Pro2_PT100 anti-phospho-Tyr ratiometric to RFU from        Pro14_anti-VEGFR2 from assay wells using biotin_anti-VEGFR2.

Results of the assays are illustrated in FIGS. 7B-D. FIG. 7B shows thequantity of VEGFR2-VEGFR2 homodimers increases on HUVEC cells withincreasing concentrations of VEGF at a 5 min stimulation time. FIG. 7Cshows VEGFR2-VEGFR2 homodimers form by 1 minute of VEGF stimulation, andremain after up to 14 minutes of stimulation. However VEGFR2phosphorylation is transient, peaking at 1 minute of stimulation anddecreasing to background level by 5 minutes.

FIG. 7D shows the quantity of VEGFR2-VEGFR2 homodimers and VEGFR2phosphorylation increases on HUVEC cells with increasing concentrationsof VEGF at a 1 min stimulation time.

Example 5 Analysis of Cell Lysates for Tie2 Homodimerization

In this example, Tie2-Tie2 homodimers were measured in cell lysates fromseveral cell lines after treatment with various concentrations ofangiopoietin-1 (Ang-1). Measurements were made using three bindingcompounds and a cleaving probe as described below.

Sample Preparation:

-   -   1. Serum-starve human umbilical vein endothelial cell line        (HUVEC) culture overnight before use.    -   2. Stimulate cell lines with 200 ng/mL Ang-1 in culture media        for a set period of time (10, 30 minutes) at 37° C.    -   3. Aspirate culture media, transfer onto ice, and add lysis        buffer to lyse cells in situ.    -   4. Scrape and transfer lysate to microfuge tube. Incubate on ice        for 30 min. Microfuge at 14,000 rpm, 4° C., for 10 min.        (Centrifugation is optional.)    -   5. Collect supernatants as lysates and aliquot for storage at        −80° C. until use.

Assay:

Assay design: As illustrated diagrammatically in FIG. 4C, Tie2-Tie2homodimers are quantified ratiometrically based on the binding ofcleaving probe (134) and binding compounds (130), and (132). Aphotosensitizer indicated by “PS” is attached to cleaving probe (134)via an avidin-biotin linkage, and binding compounds (130), and (132) arelabeled with molecular tags Pro10, and Pro14, respectively.

The total assay volume was 40 ul. The lysate volume was adjusted to 30ul with lysis buffer. The antibodies were diluted in lysis buffer up to10 ul. Typically ˜5000 to 15000 cell-equivalent of lysates was used perreaction. The detection limit was ˜1000 cell-equivalent of lysates.

-   Procedure: Final concentrations of pre-mixed binding compounds (i.e.    molecular tag- or biotin-antibody conjugates) in reaction:

Pro10_anti-Tie2: 0.25 ug/ml (Tie2 homodimer)

Pro14_anti-Tie2: 0.25 ug/ml (total Tie2)

Biotin_anti-Tie2: 0.25 ug/ml

-   -   1. To assay 96-well, add 10 ul antibody mix to 30 ul lysate and        incubate for 1 hour at RT.    -   2. Add 2 ul streptavidin-derivatized cleaving probe (final 2        ug/well) to assay well and incubate for 45 min.    -   3. Add 150 ul of wash buffer to 96-well filter plate (Millipore        MAGVN2250) and incubate for 1 hr at RT for blocking.    -   4. Empty filter plate by vacuum suction. Transfer assay        reactions to filter plate and apply vacuum to empty.    -   5. Add 200 ul wash buffer and apply vacuum to empty. Repeat one        time.    -   6. Add 200 ul illumination buffer and apply vacuum to empty.        Repeat one time.    -   7. Add 30 ul illumination buffer and illuminate for 20 min.    -   8. Transfer 10 ul of each reaction to CE assay plate for        analysis using an ABI3100 CE instrument with a 22 cm capillary        (injection conditions: 5 kV, 75 sec, 30° C.; run conditions: 425        sec, 30° C.).

-   Assay buffers are as previously described.

Data Analysis:

-   -   1. Normalize relative fluorescence units (RFU) signal of each        molecular tag against CE reference standard A315 (a        fluorescein-derivatized deoxyadenosine monophosphate that has        known peak position relative to molecular tags from the assay        upon electrophoretic separation).    -   2. Subtract RFU of “no lysate” background control from        corresponding molecular tag signals.    -   3. Report homodimerization of Tie2 as the corresponding RFU        ratiometric to RFU from Pro14_anti-Tie2 from assay wells using        biotin_anti-Tie2.

Results of the assays are illustrated in FIGS. 8A and 8B. FIG. 8A showsthe quantity of Tie2-Tie2 homodimers increase on HUVEC cells with 200ng/mL of Ang-1 at 10 min of stimulation, and decreases from that levelafter 30 min Ang-1 stimulation. FIG. 8B shows that the total amount ofTie2 receptor on the HUVEC cell does not change with Ang-1 treatment.

Example 6 Analysis of Cell Lysates for VEGFR2 After Endothelial CellIsolation Sample Preparation:

-   -   1. Magnetic beads (Dynal M450 IgG_(1,) Dynal AS, Oslo, Norway)        were conjugated to a monoclonal antibody for CD146, MCAM, an        endothelial cell surface junction protein.    -   2. HUVEC are removed from cell culture flask by trypsin-EDTA        treatment and re-suspended in PBS containing 0.1% BSA.    -   3. U937 (human histiocytic lymphoma cell line) cells in culture        are spun down in a centrifuge and re-suspended in PBS containing        0.1% BSA.    -   4. Mixtures of HUVEC and U-937 were made with a final number of        cells being 2,000,000 cells/mixture. The percent concentration        of HUVEC to U-937 respectively varied from 100/0, 90/10, 75/25,        50/50, 25/75, 10/90, 1/99, 0.1/99.9, 0.01/99.99, 0.001/99.999,        0/100.    -   5. Half of each of the cell mixtures (1,000,000 cells) were        simply spun in a microfuge and lysed using previously described        protocol in 100 uL freshly made lysis buffer.    -   6. The CD 146-conjugated beads are added to the other half of        cell mixtures at 750 ug/mL. The cells are then incubated for 30        min at 4° C. rotating gently.    -   7. Unbound cells are removed using magnetic separation using        Dynal MPC-L magnet.    -   8. Bound cells are washed 4 times with PBS containing 0.1% BSA.    -   9. After the 4^(th) wash resuspend cells in 100 uL freshly made        lysis buffer and lysed using previously described protocol.    -   10. Bicinchoninic acid (BCA) (Pierce cat #23225) is used to        quantify the protein concentration of each lysate.

Assay:

-   Assay design: VEGFR2 receptors are quantified ratiometrically based    on the binding of cleaving probe and a binding compound. A    photosensitizer indicated by “PS” is attached to cleaving probe via    an avidin-biotin linkage, and the binding compound is labeled with    molecular tag Pro14.-   The total assay volume was 40 ul. The lysate volume was adjusted to    30 ul with lysis buffer. The antibodies were diluted in lysis buffer    up to 10 ul. Typically ˜5000 to 15000 cell-equivalent of lysates was    used per reaction. The detection limit was ˜1000 cell-equivalent of    lysates.-   Procedure: Final concentrations of pre-mixed binding compounds (i.e.    molecular tag- or biotin-antibody conjugates) in reaction:

Pro14_anti-VEGFR2: 0.25 ug/ml (total VEGFR2)

Biotin_anti-VEGFR2: 0.25 ug/ml

-   -   1. To assay 96-well, add 10 ul antibody mix to 30 ul lysate and        incubate for 1 hour at RT.    -   2. Add 2 ul streptavidin-derivatized cleaving probe (final 2        ug/well) to assay well and incubate for 45 min.    -   3. Add 150 ul of wash buffer to 96-well filter plate (Millipore        MAGVN2250) and incubate for 1 hr at RT for blocking.    -   4. Empty filter plate by vacuum suction. Transfer assay        reactions to filter plate and apply vacuum to empty.    -   5. Add 200 ul wash buffer and apply vacuum to empty. Repeat one        time.    -   6. Add 200 ul illumination buffer and apply vacuum to empty.        Repeat one time.    -   7. Add 30 ul illumination buffer and illuminate for 20 min.    -   8. Transfer 10 ul of each reaction to CE assay plate for        analysis using an ABI3100 CE instrument with a 22 cm capillary        (injection conditions: 5 kV, 75 sec, 30° C.; run conditions: 425        sec, 30° C.).

-   Assay buffers are as previously described:

Data Analysis:

-   -   1. Normalize relative fluorescence units (RFU) signal of each        molecular tag against CE reference standard A315 (a        fluorescein-derivatized deoxyadenosine monophosphate that has        known peak position relative to molecular tags from the assay        upon electrophoretic separation).    -   2. Subtract RFU of “no lysate” background control from        corresponding molecular tag signals.

-   Results of the assays are illustrated in FIGS. 9A and 9B. FIG. 9A    shows the protein concentration of the lysates. The data shows a    clear trend of lower protein concentrations correlating with higher    concentrations of U-937 cells in the isolated cell lysates.    Conversely the higher concentration of HUVEC in the mixture results    in a higher protein concentration in the lysates made from cells    that have been immunogenically isolated. Lysates that have not been    magnetically isolated show no change in protein concentration, as    expected. FIG. 9B shows the presence of VEGFR2 receptors in the cell    mixtures in 5 ug of lysate. The 100/0 and 0/100 HUVEC/U-937 columns    clearly show that VEGFR2 is only expressed in HUVEC cells, and not    in U-937 cells. This ensures that the VEGFR2 seen in other mixtures    come exclusively from HUVEC. Isolation of endothelial cells enhances    the sensitivity of VEGFR2 assay from 50,000 cells/assay to    2,000-5,000 cells/assay. This is a 10-25 fold increase in    sensitivity.

Example 7 Analysis of Cell Lysates for Erk, BAD, and RSK Phosphorylation

As shown in FIG. 3, in response to VEGF, phosphorylation of variousproteins downstream of the VEGFR2 pathways (e.g., MEK, Erk, RSK, BAD,and Akt) is increased. In this example, Erk, BAD and RSK phosphorylationstates were measured in cell lysates from several cell lines aftertreatment with various concentrations of vascular endothelial growthfactor (VEGF). Measurements were made using two binding compounds and acleaving probe for each target protein as described below.

Sample Preparation:

-   -   1. Serum-starve human umbilical vein endothelial cell line        (HUVEC) culture overnight before use.    -   2. Stimulate cell lines with VEGF in culture media for a set        period of time (1, 3, 5, 9, 14 minutes) at 37° C. Exemplary        doses of VEGF are 0, 2, 10, 25, 50, 250, 1000 ng/mL.    -   3. Aspirate culture media, transfer onto ice, and add lysis        buffer to lyse cells in situ.    -   4. Scrape and transfer lysate to microfuge tube. Incubate on ice        for 30 min. Microfuge at 14,000 rpm, 4° C., for 10 min.        (Centrifugation is optional.)    -   5. Collect supernatants as lysates and aliquot for storage at        −80° C. until use.

Assay:

-   Assay design: Erk, BAD and RSK phosphorylation states are quantified    ratiometrically based on the binding of a cleaving probe and two    binding compounds. A photosensitizer is attached to the cleaving    probe via an avidin-biotin linkage, and two binding compounds are    labeled with molecular tags Pro44 and Pro32 for targeting Erk, Pro52    and Pro46 for targeting BAD, or Pro36 and Pro53 for targeting RSK.-   The total assay volume was 40 ul. The lysate volume was adjusted to    30 ul with lysis buffer. The antibodies were diluted in lysis buffer    up to 10 ul. Typically ˜5000 to15000 cell-equivalent of lysates was    used per reaction. The detection limit was ˜1000 cell-equivalent of    lysates.-   Procedure: Final concentrations of pre-mixed binding compounds (i.e.    molecular tag- or biotin-antibody conjugates) in reaction:-   biotin_anti-Erk, 20 nM-   Pro44_anti-Erk (Total), 5 nM-   Pro32_anti-phospho-Erk (Phosphorylation), 5 nM-   biotin_anti-BAD, 20 nM-   Pro52_anti-BAD (Total), 5 nM-   Pro46_anti-Phospho-BAD (Phosphorylation), 5 nM-   Biotin_anti-RSK, 20 nM-   Pro36_anti-RSK (Total), 5 nM-   Pro53_anti-phospho-RSK (Phosphorylation), 5 nM    -   1. Prime the filter plate (Millipore MAGVN2250) with 50 μl of        Assay Buffer.    -   2. Add 20 μl of Antibody mixture.    -   3. Add 10 μl of Lysate and incubate for 1 h    -   4. Add 2 μg streptavidin-derivatized cleaving probe to each        assay well, and incubate for another 30 min.    -   5. Empty filter plate by vacuum suction.    -   6. Add 200 μl of Wash buffer and empty filter plate by vacuum        suction.    -   7. Repeat step 6 one more time.    -   8. Add 50 μl of illumination buffer containing CE standard.    -   9. Illuminate the plate for 10 min.    -   10. Transfer 20 μl to the 96 well plates and run CE using        ABI-3100.-   Assay buffers are as previously described.

Data Analysis:

-   -   1. Normalize relative fluorescence units (RFU) signal of each        molecular tag against CE reference standard A315 (a        fluorescein-derivatized deoxyadenosine monophosphate that has        known peak position relative to molecular tags from the assay        upon electrophoretic separation).    -   2. Subtract RFU of “no lysate” background control from        corresponding molecular tag signals.    -   3. Report phosphorylation of Erk as the corresponding RFU        ratiometric to RFU from Pro44_anti-Erk from assay wells using        biotin_anti-Erk.    -   4. Report phosphorylation of RSK as the corresponding RFU        ratiometric to RFU from Pro36_anti-RSK from assay wells using        biotin_anti-RSK.    -   5. Report phosphorylation of BAD as the corresponding RFU        ratiometric to RFU from Pro52_anti-BAD from assay wells using        biotin_anti-BAD.

Results of the assays are illustrated in FIGS. 10A, 10B and 10C. FIGS.10A, 10B, and 10C show that the phosphorylation state of Erk, RSK, andBAD respectively increases on HUVEC cells with increasing concentrationsof VEGF at a 5 min stimulation time. It was also found thatphosphorylation of Akt increased on HUVEC cells with increasingconcentrations of VEGF (data not shown).

Example 8 Analysis of Expression of Angiogenic Genes and Proteins inEndothelial and Cancer Cell Lines

In this example, expression of angiogenic genes and proteins inendothelial and cancer cell lines was analyzed by using a Taqman assayand an assay using releasable molecular tags similar to the one inExample 3.

Example 9 Analysis of Gene and Protein Expression of Receptors inEndothelial and Cancer Cell Lines

In this example gene and protein expression of receptors were measuredin primary endothelial cell lines and human cancer cell lines. Geneexpression data was obtained using real-time quantitative PCR (TaqmanAssay), and protein expression was determined using the bindingcompounds and cleaving probes as described below. Genes assayed wereVEGFR2, VEGFR1, Tie2, Her1, Her2, Her3, VEGFR3, PDGFRA, PDGFRB, Tie1,FGFR1, FGFR2, FGFR3, FGFR4, EphA1, EphA2, EphA3, EphA4, EphA5, EphA7,EphA8, EphB1, and EphB2. Proteins assayed were VEGFR2, VEGFR1, Tie2,Her1, Her2, and Her3.

Sample Preparation:

Primary endothelial cell lines were obtained from Cambrex Bio ScienceWalkersville, Inc. and were propagated in growth media as permanufacturer's recommendations. The endothelial cell lines were HumanUmbilical Vein Endothelial Cells (HUVEC), Human Micro-VascularEndothelial Cells, Dermal (HMVEC-d), Human Pulmonary Artery EndothelialCells (HPAEC), and Human Micro-Vascular Endothelial Cells, Lung(HMVEC-L). The cancer cell lines tested were MCF7 and SKBR3 (humanbreast cancer cells), and 22RV1 (human prostate cancer cells), and werepropagated per ATCC instructions in growth media.

RNA was isolated from the cells for gene expression profiling usingTRIzol® Reagent (Invitrogen) following manufacturer's protocol.

Cells were lysed for protein expression profiling as follows.

-   -   1. Aspirate culture media, transfer onto ice, and add lysis        buffer to lyse cells in situ.    -   2. Scrape and transfer lysate to microfuge tube. Incubate on ice        for 30 min. Microfuge at 14,000 rpm, 4° C., for 10 min.        (Centrifugation is optional.)    -   3. Collect supernatants as lysates and aliquot for storage at        −80° C. until use.

Assay:

-   Gene Expression Assay design: Pre-developed Taqman probes were    purchased from Applied BioSystems. Assays were performed on 5 ng of    total RNA as per manufacturer's recommendations. Briefly, 10×    primer-probe mix (ABI), RNA, and 2× Quantitect Probe RT-PCR Master    Mix (Qiagen), 100× Quantitect RT Mix, and H₂O were combined to a    final volume of 25 ul. Cycling conditions were 30 min at 48° C., 10    min at 95° C., and 40 cycles of 15 sec at 95° C. and 1 min at 60° C.    Samples were run on the DNA Engine Opticon (MJ Research).-   Protein Assay design: Receptors were quantified ratiometrically    based on the binding of a cleaving probe and a binding compound. A    photosensitizer is attached to the cleaving probe via an    avidin-biotin linkage, and the binding compound is labeled with    molecular tag Pro14.-   The total assay volume was 40 ul. The lysate volume was adjusted to    30 ul with lysis buffer. The antibodies were diluted in lysis buffer    up to 10 ul. Typically ˜5000 to15000 cell-equivalent of lysates was    used per reaction. The detection limit is ˜1000 cell-equivalent of    lysates.-   Procedure: Final concentrations of pre-mixed binding compounds (i.e.    molecular tag- or biotin-antibody conjugates) in reaction:

Pro11_anti-VEGFR1: 0.25 ug/ml (total VEGFR1)

Biotin_anti-VEGFR1: 0.25 ug/ml

Pro14_anti-VEGFR2: 0.25 ug/ml (total VEGFR2)

Biotin_anti-VEGFR2: 0.25 ug/ml

Pro10_anti-Tie2: 0.25 ug/ml (total Tie2)

Biotin_anti-Tie2: 0.25 ug/ml

Pro10_anti-Her1: 0.1 ug/ml (total Her1)

Biotin_anti-Her1: 2 ug/ml

Pro14_anti-Her2: 0.1 ug/ml (total Her2)

Biotin_anti-Her2: 2 ug/ml

Pro99_anti-Her3: 0.1 ug/ml (total Her3)

Biotin_anti-Her3: 2 ug/ml

-   -   1. To assay 96-well, add 10 ul antibody mix to 30 ul lysate and        incubate for 1 hour at RT.    -   2. Add 2 ul streptavidin-derivatized cleaving probe (final 2        ug/well) to assay well and incubate for 45 min.    -   3. Add 150 ul of wash buffer to 96-well filter plate (Millipore        MAGVN2250) and incubate for 1 hr at RT for blocking.    -   4. Empty filter plate by vacuum suction. Transfer assay        reactions to filter plate and apply vacuum to empty.    -   5. Add 200 ul wash buffer and apply vacuum to empty. Repeat one        time.    -   6. Add 200 ul illumination buffer and apply vacuum to empty.        Repeat one time.    -   7. Add 30 ul illumination buffer and illuminate for 20 min.    -   8. Transfer 10 ul of each reaction to CE assay plate for        analysis using an ABI3100 CE instrument with a 22 cm capillary        (injection conditions: 5 kV, 75 sec, 30° C.; run conditions: 425        sec, 30° C.).

-   Assay buffers are as previously described.

Data Analysis: Gene Expression

-   -   1. The Cycle Threshold line was set using the Opticon Monitor        software. The cycle number at which the fluorescent signal from        cleaved signal probes crosses the threshold line is known as the        Cycle Threshold (C(t)).

Protein Expression

-   -   1. Normalize relative fluorescence units (RFU) signal of each        molecular tag against CE reference standard A315 (a        fluorescein-derivatized deoxyadenosine monophosphate that has        known peak position relative to molecular tags from the assay        upon electrophoretic separation).    -   2. Subtract RFU of “no lysate” background control from        corresponding molecular tag signals.

Results of the assays are summarized in Tables 3 and 4. Gene expressionvalues were split into 3 categories: “+”, which indicates a clearpresence of a gene (C(t) of <32), “Low” which indicates a C(t) of 32-38,and “−” which indicates no gene detected (C(t) >38). Detection of aprotein that has a signal above background level is represented in thetable as a “+” and no protein detected is represented as a “−”. “NT”indicates a sample was not tested. Of interest is the expression ofVEGFR1, VEGFR2, and Tie2 (all of which are endothelial markers) inendothelial cells and not in the cancer cells.

TABLE 3 VEGFR2 VEGFR1 Tie2 Her1 Her2 Her3 Pro- Pro- Pro- Pro- Pro- Pro-VEGFR3 PDGFRA PDGFRB Tie1 Cell Line Gene tein Gene tein Gene tein Genetein Gene tein Gene tein Gene Gene Gene Gene HUVEC + + +− + + + + + + + + + − − + HMVEC-d + + Low − Low − + NT + NT + NT − Low +− HPAEC + + + − + + + NT + NT + NT Low − − + HMVEC-L + + + − + + + NT +NT − NT + − − + MCF-7 Low NT − NT − NT + + + + + + Low − − − 22RV-1 − NT− NT − NT + + + + + + Low − − − SKBR3 − NT − NT − NT + + + + + + − − − −

TABLE 4 FGFR1 FGFR2 FGFR3 FGFR4 EphA1 EphA2 EphA3 EphA4 EphA5 EphA7EphA8 EphB1 EphB2 Cell Line Gene Gene Gene Gene Gene Gene Gene Gene GeneGene Gene Gene Gene HUVEC + − Low Low − + − + Low − − Low + HMVEC-d + −Low Low − + Low Low + − − − + HPAEC + − − Low − + − + − − − Low +HMVEC-L + − − Low − + − + Low − − Low + MCF-7 + + + + + + Low + − + −Low + 22RV-1 + − + + + + + Low Low + − − Low SKBR3 + + + + + + − + − − −− Low

Example 10 Parallel Analysis of VEGFR2 Homodimerization in Cell Lysateand FFPE Samples

In this example, a parallel analysis of VEGFR2-VEGFR2 homodimers stateswere measured in cell lysates and formalin-fixed paraffin embedded(“FFPE”) samples from the same treated and untreated human umbilicalvein endothelial cells (HUVEC). This results presented hereindemonstrate that VEGFR2 homodimers can be detected in cell lysate orFFPE sample format. Measurement was done following the protocoldescribed below.

Sample Preparation:

-   -   1. Seed 6e+6 human umbilical vein endothelial cell line (HUVEC)        in 15 cm cell culture dish. Seed a total of 22 plates.    -   2. Serum-starve human umbilical vein endothelial cell line        (HUVEC) culture overnight before use. 2 plates will be used for        harvesting stimulated and unstimulated cell lysate. 20 plates        will be used for harvesting stimulated and unstimulated cell        pellet for FFPE block.    -   3. Stimulate cell line with either 0 ng/ml or 500 ng/ml of VEGF        in 1.5% FBS culture media for 1 minute. Stimulate one plate at a        time.    -   4. Aspirate culture media, transfer onto ice, wash cells with 20        ml 1×PBS. For cell lysate, add 1 ml of lysis buffer to lyse        cells in situ.    -   5. Scrape and transfer lysate to microfuge tube. Incubate on ice        for 30 min. Microfuge at 14,000 rpm, 4° C., for 10 min.    -   6. Collect supernatants as lysates and aliquot for storage at        −80° C. until use.    -   7. For FFPE block, aspirate culture media, transfer onto ice,        wash cells with 20 ml 1×PBS and add 1 ml of 10% neutral buffered        formalin to cells.    -   8. Scrape and transfer cells to 15 ml tube. Combine all        stimulated HUVEC in one tube and unstimulated HUVEC in another        tube. Formalin fixed cells at 4° C. for 16 hours. Spin cell at        3000rpm at 4° C. for 5 min prior to FFPE block processing step.    -   9. Use Tissue-Tek automatic processor for processing the sample.        Hydrate each formalin fixed sample using a series of alcohols        with increasing concentration followed by treatment with        Clear-rite (Xylene substitute) and paraffin.    -   10. Embed processed samples in a paraffin block using paraffin        embedding station.    -   11. Cut 7 micron section and place on positively-charged glass        slide, air dry at RT for 1 hour and heat slide in dry oven at        60° C. for 1 hour.

Assay:

Lysate assay: Assay performed as illustrated in example 4.

FFPE assay:

-   -   1. Treat FFPE sections with 2× xylene, 2×100% ethanol, 2×70%        ethanol, 2× water to remove the paraffin and rehydrate the        sample.    -   2. Treat hydrated sections with protease 1 for 4 minutes at        37° C. to retrieval the epitope.    -   3. Block each section with 50u1 of 1.5% BSA blocking buffer for        1 hour. Aspirate blocking buffer, add 30 ul of antibody mix to        each section and incubation overnight at 4° C. with shaking.    -   4. Wash each section with 50 ul of 1×PBST (1×PBS with 0.25%        Triton-x-100) twice and follow by 1×PBS.    -   5. Aspirate 1×PBS, incubate each section with 30 ul of 2.5 ug/ml        of streptavidin-scissor mix for 1 hour in dark.    -   6. Rinse slides with 3× water. Add 30 ul of illumination buffer        to each section and illuminate for 2 hours at 4° C.    -   7. Following illumination, incubate slides are RT for 1 hour.    -   8. Remove illumination buffer from each section and analyze        sample on CE instrument.        antibody conjugates in reaction:

Pro10_anti-VEGFR2: 2 ug/ml (VEGFR2 homodimer)

Biotin_anti-VEGFR2: 2 ug/ml (VEGFR2 homodimer)

Results of the comparison between lysate and FFPE assays are shown inFIG. 12. In the absence of VEGF stimulation, only basal levels ofdimerization were detected in both formats. With VEGF stimulation bothassays successfully detected a large increase in VEGFR2homodimerization.

Example 11 Analysis of Xenograft Tissue Lysates for Total VEGFR2Receptor and Homodimerization

In this example, total VEGFR2 and VEGFR2 homodimerization was measuredin mouse-human xenograft tissue created using a variety of human celllines, including DU145, H522, H441, BT474, CALU-3, A549, MDA-MB-231,A431, H460, OVCAR-3 and HCT116. As controls, lysates of unstimulated andVEGF-stimulated HUVEC cultures were prepared. Measurements were madeusing two binding compounds and a cleaving probe as described below.

Because xenograft lysates potentially contained human and mouse forms ofVEGFR2, an ELISA was performed to confirm that the VEGFR2 detected inthis assay came from the human cells, and not mouse cells. It would beexpected that the anti-VEGFR2 antibodies used here would react withhuman but not mouse VEGFR2 as the antibodies were raised in mice againstrecombinant human VEGFR2. In a 96-well plate, dilutions of recombinantmouse VEGFR2/Fc chimera (R&D Systems) or human VEGFR2/Fc chimera (R&DSystems) were made from 200 ng to 0.3 ng per well in phosphate bufferedsaline (PBS). Extra wells were included as protein-free controls. Theproteins were allowed to adsorb for 1 hour at room temperature. Theplates were blocked for 1 hour with 0.05% Tween 20 in PBS and thenprobed with the same 1 ug/ml Biotin_anti-VEGFR2 as used above. Followingwashes with 0.05% Tween 20 in PBS, retained Biotin_anti-VEGFR2 wasdetected with streptavidin-linked horseradish peroxidase followed byapplication of a chromogenic substrate that absorbs light at 460 nm. Asshown in FIG. 12A, the cleaving probe was specific to human VEGFR2.

Control Sample Preparation:

-   -   1. Serum-starve human umbilical vein endothelial cell line        (HUVEC) culture overnight before use.    -   2. Stimulate cell lines for 1 minute with media pre-warmed to        37° C. containing either or 200 ng/mL VEGF or no VEGF.    -   3. Aspirate culture media, transfer onto ice, and add cold lysis        buffer to lyse cells in situ.    -   4. Scrape and transfer lysate to microfuge tube. Incubate on ice        for 30 minutes.    -   5. Centrifuge at 14,000 rpm, 4° C., for 10 minutes.    -   6. Collect supernatants as lysates and aliquot for storage at        −80° C. until use.        Tissue preparation:    -   1. Finely mince tissue using razor blades.    -   2. Mix with 0.5-1 ml lysis buffer per 1 g tissue.    -   3. Further disrupt tissue with homogenizer using brief pulses.    -   4. Incubate on ice for 30 minutes.    -   5. Centrifuge at 14,000 rpm, 4° C., for 10 minutes.    -   6. Collect supernatants as lysates and aliquot for storage at        −80° C. until use.

Assay:

The assay design was similar to that of Example 4, illustrated in FIG.7A. VEGFR2-VEGFR2 (R2-R2) homodimers (900) were quantified based on thebinding of a cleaving probe (902), a first binding compound (904) whichwas specific for the same antigenic determinant on VEGFR2 as thecleaving probe and a second binding compound (906) which was specificfor a different antigenic determinant on VEGFR2 from the cleaving probe.A photosensitizer was attached to the cleaving probe (902) via anavidin-biotin linkage, and the first and second binding compounds werelabeled with molecular tags Pro10 and Pro14, respectively.

The assay was run on serial dilutions of the lysate, typically from ˜50ug protein to ˜0.5 ug plus a zero-protein control. Protein in eachlysate sample was determined by a bicinchoninic assay (PierceBiotechnology) against an albumin standard. The total assay volume was40 ul. The lysate volume was adjusted to 30 ul with lysis buffer. Theantibodies were diluted in lysis buffer to 5 ul. Thestreptavidin-derivatized cleaving probe was also diluted in lysis bufferto 5 ul.

-   Procedure: Final concentrations of pre-mixed binding compounds (i.e.    molecular tag- or biotin-antibody conjugates) in reaction:

Pro10_anti-VEGFR2: 1.0 ug/ml (VEGFR2 homodimer)

Pro14_anti-VEGFR2: 1.0 ug/ml (total VEGFR2)

Biotin_anti-VEGFR2: 1.0 ug/ml

-   -   9. In a 96-well assay plate, add 5 ul antibody mix to each 30 ul        lysate and incubate for 1 hour at RT.    -   10. Add 5 ul streptavidin-derivatized cleaving probe (final 2.5        ug/well) to assay well and incubate for 45 min.    -   11. Add 150 ul of wash buffer to 96-well filter plate (Millipore        MAGVN2250) and incubate for 1 hr at RT for blocking.    -   12. Empty filter plate by vacuum suction. Transfer assay        reactions to filter plate and apply vacuum to empty.    -   13. Add 200 ul wash buffer and apply vacuum to empty. Repeat one        time.    -   14. Add 200 ul illumination buffer and apply vacuum to empty.        Repeat one time.    -   15. Add 30 ul illumination buffer and illuminate for 20 min.    -   16. Transfer 10 ul of each reaction to CE assay plate for        analysis using an ABI3100 CE instrument with a 22 cm capillary        (injection conditions: 5 kV, 75 sec, 30° C.; run conditions: 425        sec, 30° C.).

-   Lysis buffer composition and data analysis were identical to Example    4.

Various xenograft tissues were screened for total VEGFR2-VEGFR2homodimers. Results of the xenograft screening are shown in FIG. 13B.Cell lines used to make the xenografts labeled A-K refer to cell linesDU145, H522, H441, BT474, CALU-3, A549, MDA-MB-231, A431, H460, OVCAR-3and HCT116, respectively. The levels of VEGFR2 expression anddimerization varied greatly over the cell lines tested, with H441 givingthe highest signals.

These results demonstrate that VEGFR2 dimers can be detected in samplesobtained from tissues. In addition, these results indicate that thistype of assay can be used in identifying cell lines for xenograph modelsthat are useful in testing anti-angiogenic drug susceptibility. By FIG.13B, cell line H441 can, for example, be useful in this regard. Further,this type of analysis can be useful in screening representative celllines of various cancer types to identify classes of cancers that areparticular susceptible to anti-angiogenic therapy. Such assays can alsobe used as part of diagnostics for assessing or predicting patientresponsiveness to anti-angiogenic therapy.

Example 12 Analysis of Lung Cancer Tissue Lysates for Total VEGFR2Receptor and Homodimerization

In this example, total VEGFR2 and VEGFR2 homodimerization was measuredin normal and cancerous lung tissue. Preparation of tissue lysates wasidentical to the preparation procedure of Example 11. The assay methodsand analysis were also identical except that the HUVEC control cellswere stimulated for 2 minutes.

As shown in FIG. 14, lung cancer tissues were found to have a widevariety of total VEGFR2, homodimer levels and ratios of homodimer tototal receptor, while the normal tissue samples tested showed low levelsof VEGFR2 expression and dimerization.

These results demonstrate that VEGFR2 dimers can be detected in samplesobtained from human tumor tissues. Further, this assay illustrates themeasurement of a marker that can be indicative of anti-angiogenic drugsusceptibility. Assay employing this marker and others associated withthe angiogenic state can be utilized in predicting clinical outcomes fora variety of anti-angiogenic therapies. This type of assay may also beused as a surrogate marker for response to anti-angiogenic and otheranti-cancer therapy.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

1. A method for detecting activation of endothelial cells in a testsample, comprising: measuring the level of a protein complex in theendothelial cells in a test sample, wherein the protein complex isformed between a first cellular component and a second cellularcomponent that are cellular components in an angiogenesis signalingpathway; and wherein a difference in the level of the protein complexrelative to the level of the protein complex in a reference sampledetects activation of endothelial cells in the test sample.
 2. Themethod of claim 1, wherein the first cellular component and the secondcellular component are each independently selected from the groupconsisting of VEGFR, Nrp, heparin sulphate, VE-cadherin, Tie, VEGF,P1GF, PDGFR, EphA, EphB, Flt, FGFR, Stat, BAD, RSK, P13K, FAK, Src,P70S6K, SHC, SHC, Akt, Erk, JNK, P38, and MEK.
 3. The method of claim 1,wherein the said protein complex is selected from the group consistingof VEGFR1 homodimers, VEGFR2 homodimers, VEGFR1-VEGFR2 heterodimers,VEGFR2-VEGFR3 heterodimers, VEGFR2-SHC complexes, and VEGFR3-SHCcomplexes.
 4. The method of claim 1, wherein the test sample containscirculating endothelial cells or circulating endothelial cellprogenitors.
 5. The method of claim 4, further comprising isolatingcirculating endothelial cells or circulating endothelial cellprogenitors.
 6. The method of claim 5, wherein circulating endothelialcells or circulating endothelial cell progenitors are isolated byimmunomagnetic isolation.
 7. The method of claim 1, wherein the testsample contains tumor endothelium.
 8. The method of claim 1, wherein thetest sample is a blood sample.
 9. The method of claim 1, wherein thetest sample is a fixed tissue sample.
 10. The method of claim 1, whereinthe step of measuring the level of the protein complex in theendothelial cells comprises: mixing (i) the test sample; (ii) a cleavingprobe, which is capable of binding the first cellular component and hasa cleavage-inducing moiety with an effective proximity; and (iii) one ormore binding compounds, wherein each of the binding compounds is capableof binding the first or second cellular component and wherein each ofthe one or more binding compounds has one or more molecular tags eachattached thereto by a cleavable linkage; wherein cleavage of thecleavable linkage(s) within the effective proximity of thecleaving-inducing moiety of the cleaving probe releases the moleculartag(s), wherein detecting the released molecular tag(s) provides ameasurement of the protein complex.
 11. The method of claim 10, whereinthe first or second cellular component is a cell surface receptor. 12.The method of claim 11, wherein the cell surface receptor is selectedfrom the group consisting of VEGFR, Tie, PDGFR, and FGFR.
 13. The methodof claim 11, wherein the cell surface receptor is VEGFR2 or Tie-2. 14.The method of claim 10, wherein the molecular tag(s) attached todifferent binding compounds each have a different separationcharacteristics.
 15. The method of claim 14, further comprisingseparating the released molecular tag(s).
 16. The method of claim 10,wherein said step of mixing includes generating an active species bysaid cleavage-inducing moiety, wherein the active species cleaves saidcleavable linkages within said effective proximity.
 17. The method ofclaim 10, further comprising measuring the level of an effector proteinin the angiogenesis signaling pathway that has a post-translationalmodification site in the endothelial cells in the test sample.
 18. Themethod of claim 17, wherein the effector protein is selected from thegroup consisting of Stat, BAD, RSK, P13K, FAK, Src, P70S6K, SHC, Akt,Erk, JNK, P38, and MEK.
 19. The method of claim 1, wherein the testsample is obtained from an individual who is suspected of having adisease associated with undesirable angiogenesis, and wherein detectingactivation of endothelial cells in the test sample indicates that theindividual has the disease.
 20. A method for screening patients todetermine the likelihood that a patient will respond to treatment by ananti-angiogenic agent, comprising: measuring the level of a proteincomplex in the endothelial cells in a test sample from a patient,wherein the protein complex is formed between a first cellular componentand a second cellular component that are cellular components in anangiogenesis signaling pathway; wherein an increase in the level of theprotein complex in the test sample from the patient relative to areference level characteristic of normal endothelial cells indicatesthat the likelihood that the patient will respond to treatment by ananti-angiogenic agent.
 21. A method for determining whether a patientwill respond to treatment by an anti-angiogenic agent, comprising:measuring the level of a protein complex in the endothelial cells in atest sample from a patient who has been treated with an anti-angiogenicagent, wherein the protein complex is formed between a first cellularcomponent and a second cellular component that are cellular componentsin an angiogenesis signaling pathway; wherein a decrease in the level ofthe protein complex in the vascular (or CEC/CECP) endothelial cells fromthe test sample relative to a reference level of the protein complex inendothelial cells in the patient prior to the treatment, indicates thatthe patient is likely to respond to the treatment by the anti-angiogenicagent.
 22. A method for determining whether a patient has developedresistance to treatment of an anti-angiogenic agent, comprising:measuring the level of a protein complex in the endothelial cells in atest sample from a patient who has been treated with an anti-angiogenicagent, wherein the protein complex is formed between a first cellularcomponent and a second cellular component that are cellular componentsin an angiogenesis signaling pathway; wherein an increase in the levelof the protein complex in the endothelial cells from the test samplerelative to a reference level of the protein complex in the endothelialcells in the patient prior to treatment, indicates that the patient haslikely developed resistance to the treatment of the anti-angiogenicagent.
 23. A method for detecting activation of endothelial cells in atest sample, comprising: measuring in a test sample the levels of two ormore different cellular components that participate in one or moreangiogenesis signaling pathways; wherein a difference in the levels ofthe two or more different cellular components relative to referencelevels of the two or more different cellular components, indicatesactivation of endothelial cells in a test sample.
 24. The method ofclaim 23, wherein the two or more cellular component are selected fromthe group consisting of VEGFR, Nrp, heparin sulphate, VE-cadherin, Tie,VEGF, P1GF, PDGFR, EphA, EphB, Flt, FGFR, Stat, BAD, RSK, P13K, FAK,Src, P70S6K, SHC, SHC, Akt, Erk, JNK, P38, and MEK.
 25. The method ofclaim 23, wherein the test sample contains circulating endothelial cellsor circulating endothelial cell progenitors.
 26. The method of claim 23,wherein the test sample contains tumor endothelium.
 27. The method ofclaim 23, wherein the test sample is a blood sample.
 28. The method ofclaim 23, wherein the test sample is a fixed tissue sample.
 29. Themethod of claim 23, wherein the step of measuring the levels of two ormore different cellular components in the endothelial cells includesmixing (i) the test sample; (ii) a cleaving probe, which is capable ofbinding one of the two or more cellular components and has acleavage-inducing moiety with an effective proximity; and (iii) one ormore binding compounds, wherein each of the two or more cellularcomponents is bound by at least one member of the one or more bindingcompounds, and wherein each of the binding compounds has one or moremolecular tags each attached thereto by a cleavable linkage; whereincleavage of the cleavable linkage(s) within the effective proximity ofthe cleaving-inducing moiety of the cleaving probe releases themolecular tag(s), wherein detecting the released molecular tag(s)provides a measurement of the levels of two or more different cellularcomponents in the endothelial cells.
 30. The method of claim 29, whereinat least one of the two or more cellular components is a cell surfacereceptor.
 31. The method of claim 30, wherein the cell surface receptoris selected from the group consisting of VEGFR, Tie, PDGFR, and FGFR.32. The method of claim 30, wherein the cell surface receptor is VEGFR2or Tie-2.
 33. The method of claim 30, wherein the molecular tag(s)attached to different binding compounds each have a different separationcharacteristics.
 34. The method of claim 33, further comprisingseparating the released molecular tag(s).
 35. The method of claim 30,wherein said step of mixing includes generating an active species bysaid cleavage-inducing moiety, wherein the active species cleaves saidcleavable linkages within said effective proximity.
 36. The method ofclaim 30, further comprising measuring the level of an effector proteinin the angiogenesis signaling pathway that has a post-translationalmodification site in the endothelial cells in the test sample.
 37. Themethod of claim 36, wherein the effector protein is selected from thegroup consisting of Stat, BAD, RSK, P13K, FAK, Src, P70S6K, SHC, Akt,Erk, JNK, P38, and MEK.